HIV-1 vaccines, antibody compositions related thereto, and therapeutic and prophylactic uses thereof

ABSTRACT

The subject invention provides a recombinant nucleic acid molecule which encodes a mutant HIV-1 gp120 envelope glycoprotein comprising a V3 loop deletion and a C4 domain.sub.(W→X) point mutation wherein X is an amino acid residue other than tryptophan, and the HIV-1 gp120 envelope glycoprotein encoded thereby. 
     The subject invention further provides a method of obtaining partially purified antibodies which specifically bind to the CD4-binding domain of HIV-1 gp120 envelope glycoprotein, the partially purified antibodies produced thereby, and pharmaceutical compositions comprising same. Finally, the subject invention provides methods of treating HIV-1-infected subjects, and of reducing the likelihood of HIV-1-exposed and non-HIV-1-exposed subjects&#39; becoming infected with HIV-1 using the pharmaceutical compositions of the subject invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

The life cycle of animal viruses is characterized by a series of events that are required for the productive infection of the host cell. The initial step in the replicative cycle is the attachment of the virus to the cell surface, which attachment is mediated by the specific interaction of the viral attachment protein (VAP) to receptors on the surface of the target cell. The differential pattern of expression of these receptors is largely responsible for the host range and tropic properties of viruses. In addition, an effective immune response against many viruses is mediated through neutralizing antibodies directed against the VAP. The interaction of the VAP with cellular receptors and the immune system therefore plays a critical role in infection and pathogenesis of viral disease.

The human immunodeficiency virus type 1 (HIV-1) infects primarily helper T lymphocytes, dendritic cells, and monocytes/macrophages--cells that express surface CD4--leading to a gradual loss of immune function. This loss of function results in the development of the human acquired immunodeficiency syndrome (AIDS) (1). The initial phase of the HIV-1 replicative cycle involves the high-affinity interaction between the HIV-1 exterior envelope glycoprotein gp120 and cell surface CD4 (K_(d) approximately 4×10⁻⁹ M) (2). Several lines of evidence demonstrate the requirement of this interaction for viral infectivity. The introduction into CD4⁻ human cells of cDNA encoding CD4 is sufficient to render otherwise resistant cells susceptible to HIV-1 infection (3). In vivo, viral infection appears to be restricted to cells expressing CD4, indicating that the cellular tropism of HIV-1 is largely determined by the pattern of cellular expression of CD4. Following the binding of HIV-1 gp120 to cell surface CD4, viral and target cell membranes fuse by a mechanism that is poorly understood, resulting in the introduction of the viral capsid into the target cell cytoplasm (4).

Mature CD4 has a relative molecular mass (Mr) of 55 kDa and consists of an N-terminal 372-amino acid extracellular domain containing four tandem immunoglobulin-like regions (V1-4), followed by a 23-amino acid transmembrane domain and a 38-amino acid cytoplasmic segment (5, 6). In experiments using truncated sCD4 proteins, it has been shown that the determinants for high-affinity binding to HIV-1 gp120 lie solely within the N-terminal immunoglobulin-like domain (V1) (7-9). Mutational analysis of V1 has defined a discrete binding site (residues 38-52) that comprises a region structurally homologous to the second complementarity-determining region (CDR2) of immunoglobulin genes (9).

The production of large quantities of sCD4 has permitted a structural analysis of the two N-terminal immunoglobulin-like domains (V1V2). The structure determined at 2.3 angstrom resolution reveals that the molecule has two tightly-associated domains, each of which contains the immunoglobulin-fold connected by a continuous beta strand. The putative binding sites for monoclonal antibodies, class II major histocompatibility complex (MHC) molecules, and HIV-1 gp120, as determined by mutational analyses, map on the molecular surface (10, 11).

The HIV-1 envelope gene env encodes an envelope glycoprotein precursor, gp160, which is cleaved by cellular proteases before transport to the plasma membrane to yield gp120 and gp41. The membrane-spanning glycoprotein, gp41, is non-covalently associated with gp120, a purely extracellular glycoprotein. The mature gp120 molecule is heavily glycosylated (approximately 24 N-linked oligosaccharides), contains approximately 480 amino acid residues with 9 intra-chain disulfide bonds (12), and projects from the viral membrane as a dimeric or multimeric molecule (13).

Mutational studies of HIV-1 gp120 have delineated important functional regions of the molecule. The regions of gp120 that interact with gp41 map primarily to the N- and C-termini (14). The predominant strain-specific neutralizing epitope on gp120 is located in the 32-34 amino acid residue third variable loop, herein referred to as the V3 loop, which resides near the center of the gp120 sequence (15). The CD4 binding site maps to discontinuous regions of gp120 that include highly conserved or invariant amino acid residues in the second, third, and fourth conserved domains (the C2, C3, and C4 domains) of gp120 (16). It has been postulated that a small pocket formed by these conserved residues within gp120 could accommodate the CDR2 loop of CD4, a region defined by mutational analyses as important in interacting with gp120 (17).

HIV-1 gp120 not only mediates viral attachment to surface CD4 molecules, but also serves as the major target of antibodies which neutralize non-cell-associated virus and inhibit cell to cell viral transmission.

There are two major classifications of HIV-1-neutralizing antibodies: type-specific and group-common (15). Type-specific neutralizing antibodies primarily recognize linear determinants in the highly variable V3 loop of gp120. These antibodies act by inhibiting fusion between HIV-1 and the target cell membrane, and generally neutralize only a particular isolate of, or closely related strains of, HIV-1. Sequence variation within the V3 loop, as well as outside of this region, permits viruses to escape neutralization by anti-V3 loop antibodies. In contrast, group-common neutralizing antibodies primarily recognize discontinuous or conformational epitopes in gp120, and possess the ability to neutralize a diverse range of HIV-1 isolates. These broadly neutralizing antibodies often recognize a site on gp120 which overlaps the highly conserved CD4-binding site, and thus inhibits gp120-CD4 binding.

A structural relationship has been demonstrated between the V3 loop and the C4 region of gp120 which region constitutes both part of the CD4 binding site and part of the conserved neutralization epitopes. It was observed that deleting the V3 loop resulted in significantly increased binding of a panel of broadly neutralizing hMoAbs (neutralizing human monoclonal antibodies) to the CD4 binding site (18).

A major goal in AIDS vaccine development is to develop a vaccine able to protect a subject against the numerous genetic variants of HIV-1 that infect humans. Although cell-mediated immune responses might serve to control infection in HIV-1-infected individuals, several lines of evidence demonstrate that protection against infection is mainly mediated by neutralizing antibodies directed against gp120. Early experiments showed that immunization of chimpanzees with recombinant gp120 induced a protective immune response against challenge with the homologous HIV-1 strain (17). This protection correlated with the presence of high-titer neutralizing antibodies against the V3 loop of gp120. In addition, passive immunization of chimpanzees with a V3-loop neutralizing monoclonal antibody resulted in protection against challenge with the homologous HIV-1 strain (19). Although protection against challenge was demonstrated in these two experiments, recent studies have questioned the clinical relevance of these findings. For example, these neutralizing antibodies recognize the V3 loop determinants of a single strain, and not conserved or discontinuous epitopes. Thus, these antibodies lack the ability to neutralize the broad spectrum of HIV-1 strains present in an HIV-1 population. Furthermore, the challenge virus was the homologous HIV-1 laboratory adapted LAI (HTLV-IIIB) strain and not one of the primary isolates that contain considerable gp120 sequence heterogeneity. Since these experiments showed that gp120 subunit vaccination induces an immune response effective against only the homogeneous HIV-1 strain used as an antigen, it is unlikely that the vaccination regimens used in these studies would be useful in humans.

Individuals infected by HIV-1 typically develop antibodies that neutralize the virus in vitro, and neutralization titers decrease with disease progression (19). Analysis of sera from HIV-1-infected humans indicates that type-specific neutralizing antibodies appear early in infection. Later in the course of infection, a more broadly neutralizing antibody response develops. However this antibody response is of significantly lower titer and/or affinity. Fractionation studies of HIV-1 antibody-positive human sera reveal that the type-specific neutralizing activity is primarily directed against linear determinants in the V3 loop of gp120 (20). There was no correlation found among antibodies between the ability to neutralize divergent HIV-1 isolates and reactivity to the V3 loop of these isolates. In contrast, the broadly neutralizing antibodies present in HIV-1 antibody-positive human sera primarly recognize discontinuous epitopes in gp120 which overlap the CD4-binding site and block gp120-CD4 binding. In other words, the broadly neutralizing activity of neutralizing antibodies is not merely the result of additive anti-V3 loop reactivities against diverse HIV-1 isolates which appear during infection.

Recently, several groups have generated human monoclonal antibodies (hMoAbs) derived from HIV-1 infected individuals which possess type-specific or group-common neutralizing activities (17). The type-specific neutralizing hMoAbs were found to recognize linear determinants in the V3 loop of gp120. In contrast, the group-common neutralizing hMoAbs generally recognize discontinuous epitopes which overlap the CD4-binding site and block gp120-CD4 binding.

The V3 loop is a highly immunodominant region of gp120 which partially interacts with the CD4-binding region. The presence of the V3 loop region on gp120 may skew the humoral immune response away from producing antibodies which specifically bind to the CD4-binding domain of gp120. Furthermore, the advantages of removing the V3 loop to expose the CD4-binding domain of gp120 to the immune system would be countered by the fact that the exposed CD4-binding site would still have a high affinity for cell surface CD4. In other words, a mutant gp120 protein missing only the V3 loop would quickly bind to CD4+ cells and would thus be hampered in generating an immune response against the exposed CD4-binding site.

The subject invention provides a mutant HIV-1 gp120 envelope glycoprotein which overcomes both the problems of V3 loop immunodominance and of the high affinity to CD4. The subject invention further provides vaccines comprising the mutant HIV-1 gp120 envelope glycoprotein, antibodies which specifically bind to the CD4-binding site of HIV-1 gp120 envelope glycoprotein, pharmaceutical compositions comprising these antibodies, and methods of using these vaccines and compositions to treat or prevent HIV-1 infection.

SUMMARY OF THE INVENTION

The subject invention provides a recombinant nucleic acid molecule which encodes a mutant HIV-1 gp120 envelope glycoprotein comprising a V3 loop deletion and a C4 domain.sub.(W→X) point mutation, wherein X is an amino acid residue other than tryptophan. In the preferred embodiment, X is a valine residue.

In one embodiment, the nucleic acid molecule is a DNA molecule. The DNA molecule may be a plasmid. In one embodiment, the plasmid comprises the sequence of the plasmid designated PPI4-tPA.

In one embodiment, the C4 domain is an HIV-1_(LAI) gp120 envelope glycoprotein C4 domain. The mutant HIV-1 gp120 envelope glycoprotein may be a mutant HIV-1_(LAI) gp120 envelope glycoprotein.

In another embodiment, the C4 domain is an HIV-1_(JR-FL) gp120 envelope glycoprotein C4 domain. The mutant HIV-1 gp120 envelope glycoprotein may be a mutant HIV-1_(JR-FL) gp120 envelope glycoprotein.

The subject invention also provides the mutant HIV-1 gp120 envelope glycoprotein encoded by the recombinant nucleic acid molecule of the subject invention.

The subject invention further provides a vaccine which comprises a therapeutically effective amount of the mutant HIV-1 gp120 envelope glycoprotein of the subject invention, and an adjuvant.

The subject invention further provides a method of treating an HIV-1-infected subject, which comprises immunizing the HIV-1-infected subject with the vaccine of the subject invention, thereby treating the HIV-1-infected subject.

The subject invention further provides a vaccine which comprises a prophylactically effective amount of the mutant HIV-1 gp120 envelope glycoprotein of the subject invention, and an adjuvant.

The subject invention further provides a method of reducing the likelihood of an HIV-1-exposed subject's becoming infected with HIV-1, which comprises immunizing the HIV-1-exposed subject with the vaccine of the subject invention, thereby reducing the likelihood of the HIV-1-exposed subject's becoming infected with HIV-1.

The subject invention further provides a method of reducing the likelihood of a non-HIV-1-exposed subject's becoming infected with HIV-1, which comprises immunizing the non-HIV-1-exposed subject with the vaccine of the subject invention, thereby reducing the likelihood of the non-HIV-1-exposed subject's becoming infected with HIV-1.

The subject invention further provides a method of obtaining partially purified antibodies which specifically bind to the CD4-binding domain of HIV-1 gp120 envelope glycoprotein, which method comprises (a) immunizing a non-HIV-1-exposed subject with the vaccine of the subject invention, (b) recovering from the immunized subject serum comprising said antibodies, and (c) partially purifying said antibodies, thereby obtaining partially purified antibodies which specifically bind to the CD4-binding domain of HIV-1 gp120 envelope glycoprotein. In the preferred embodiment, the subject is a human.

The subject invention further provides the partially purified antibodies produced by the method of the subject invention.

The subject invention further provides a pharmaceutical composition, which comprises a therapeutically effective amount of the partially purified antibodies of the subject invention, and a pharmaceutically acceptable carrier.

The subject invention further provides a method of treating an HIV-1-infected subject, which comprises administering to the subject a dose of the pharmaceutical composition of the subject invention effective to reduce the population of HIV-1-infected cells in the HIV-1-infected subject, thereby treating the HIV-1-infected subject

The subject invention further provides a method of treating an HIV-1-infected subject, which comprises administering to the subject a dose of the pharmaceutical composition of the subject invention effective to reduce the population of HIV-1 in the HIV-1-infected subject, thereby treating the HIV-1-infected subject.

The subject invention further provides a composition which comprises a prophylactically effective amount of the partially purified antibodies of the subject invention, and a pharmaceutically acceptable carrier.

The subject invention further provides a method of reducing the likelihood of an HIV-1-exposed subject's becoming infected with HIV-1, which comprises administering to the HIV-1-exposed subject a dose of the composition of the subject invention effective to reduce the population of HIV-1 in the HIV-1-exposed subject, thereby reducing the likelihood of the subject's becoming infected with HIV-1.

In one embodiment, the subject is a medical practitioner. In another embodiment, the subject is a newborn infant.

Finally, the subject invention provides a method of reducing the likelihood of a non-HIV-1-exposed subject's becoming infected with HIV-1 as a result of exposure thereto during an incident wherein there is an increased risk of exposure to HIV-1, which comprises administering to the subject immediately prior to the incident a dose of the composition of the subject invention effective to reduce the population of HIV-1 to which the subject is exposed during the incident, thereby reducing the likelihood of the subject's becoming infected with HIV-1. In one embodiment, the subject is a medical practitioner.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

gp120 structure.

Shown is a box diagram of HIV-1 gp120 depicting the boundaries of the five constant domains (C1-C5) and the five variable domains (V1-V5). The amino acid residue numbering above the box begins at the initiator methionine found at the beginning of the signal sequence (S) and is approximated based on a consensus of all known HIV-1 gp120 amino acid sequences. Also shown are the C4 domain amino acid sequences of HIV-1 strains LAI and JR-FL. Above the C4 domain sequences are indicated two mutations that reduce gp120 binding to cell surface CD4; tryptophan to valine and aspartate to alanine.

FIG. 2

PPI4-tPA-gp120_(LAI).

Expression vector with the HIV-1_(LAI) gp120 gene fused to the CMV MIE promoter, and the tPA signal sequence replacing the HIV-1 gp120 signal sequence. Abbreviations: CMV MIE=cytomegalovirus major immediate early, E=enhancer, P=promoter, EXA=Exon A, INA=Intron A, EXB=Exon B, tPA ss=human tissue plasminogen activator signal sequence, gp120=glycoprotein 120, BGH=bovine growth hormone, AMP=ampicillin resistance gene, and DHFR=dihydrofolate reductase gene.

FIG. 3

CMV MIE promoter fused to tPA-gp120_(LAI).

The nucleotide sequence (SEQ ID NO: 13) of the CMV MIE promoter/enhancer region is shown fused to the HIV-1_(LAI) gp120 gene that contains the tPA signal sequence. The numbering of nucleotide sequence begins with the HincII site and the numbering of the amino acid sequence (SEQ ID NO: 14) begins with the first methionine found in the tPA signal sequence. The tPA signal sequence is fused in-frame to Thr₃₁ of gp120, the first amino acid found in mature gp120. The signal sequence is shown in bold as are various landmark restriction sites used for cloning as discussed in the text. The locations of Exon A, Intron A, Exon B and the transcription start site and the signal cleavage site are indicated.

FIG. 4

Transient expression of gp120.

Autoradiograph of ³⁵ S-labeled supernatants from COS cell transfectants, immunoprecipitated with a CD4-immunoglobulin-Protein A-Sepharose complex, and run on a reducing 10% SDS-PAGE gel. The plasmids used for transfection were: Lane 1: Mock transfected cells; lane 2: a vector encoding a CD4-immunoglobulin chimera as a positive transfection control; lane 3: PPI4-tPA-gp120_(LAI) ; and lane 4: PPI4-tPA-gp120_(JR-FL). Positions of molecular weight markers are indicated.

FIG. 5

Determination of gp120 concentration by ELISA.

Panel A: Concentrations of gp120 in media of CHO cell lines, stably transfected with PPI4-tPA-gp120_(LAI), determined by ELISA. Panel B: A standard curve was established using known amounts of gp120.

FIG. 6

Expression of gp120 in stably transfected CHO cells.

Autoradiograph of ³⁵ S-labeled supernatants from stable CHO cell lines, immunoprecipitated with a CD4-immunoglobulin-Protein A-Sepharose complex, and run on a reducing 10% SDS-PAGE gel. Lane 1: clone 9; lane 2: clone 13; lane 3: clone 6; lane 4: Clone 5. Positions of molecular weight markers are indicated.

FIG. 7

tPA-gp120_(JR-FL).

The nucleotide (SEQ ID NO: 15) and deduced amino acid (SEQ ID NO: 16) sequence of the tPA signal sequence fused to HIV-1_(JR-FL) gp120 is shown. The NarI and NotI restriction endonuclease sites used for cloning are shown in bold. The predicted site of cleavage by signal peptidase between Arg₃₅ and Val₃₆ is indicated.

FIG. 8

tPA-gp120_(LAI) -V3(-).

The nucleotide (SEQ ID NO: 17) and deduced amino acid (SEQ ID NO: 18) sequence of the tPA signal sequence fused to HIV-1_(LAI) gp120 with the V3 loop deleted and replaced with the pentapeptide TGAGH is shown. The V3 loop replacement and the NarI and NotI restriction endonuclease sites used for cloning are shown in bold. The predicted site of cleavage by signal peptidase between Arg₃₅ and Thr₃₆ is indicated.

FIG. 9

tPA-gp120_(JR-FL) -V3(-).

The nucleotide and deduced amino acid sequence of the tPA signal sequence fused to HIV-1_(JR-FL) gp120 with the V3 loop deleted and replaced with the pentapeptide TGAGH is shown. The V3 loop replacement and the NarI and NotI restriction endonuclease sites used for cloning are shown in bold. The predicted site of cleavage by signal peptidase between Arg₃₅ and Val₃₆ is indicated.

FIG. 10

tPA-gp120_(LAI) -V3(-)-CD4(-).

Shown is the nucleotide (SEQ ID NO: 21) and deduced amino acid (SEQ ID NO: 22) sequence of the tPA signal sequence fused to HIV-1_(LAI) gp120, with the V3 loop deleted and replaced with the pentapeptide TGAGH, and Trp₄₀₈ mutated to Val. The mutations and the NarI and NotI restriction endonuclease sites used for cloning are shown in bold. The predicted site of cleavage by signal peptidase between Arg₃₅ and Thr₃₆ is indicated.

FIG. 11

tPA-gp120_(JR-FL) -V3(-)-CD4(-).

Shown is the nucleotide (SEQ ID NO: 23) and deduced amino acid (SEQ ID NO: 24) sequence of the tPA signal sequence fused to HIV-1_(JR-FL) gp120, with the V3 loop deleted and replaced with the pentapeptide TGAGH, and Trp₃₉₆ mutated to Val. The mutations and the NarI and NotI restriction endonuclease sites used for cloning are shown in bold. The predicted site of cleavage by signal peptidase between Arg₃₅ and Val₃₆ is indicated.

FIG. 12

tPA-gp120_(LAI) -CD4(-).

Shown is the nucleotide (SEQ ID NO: 25) and deduced amino acid (SEQ ID NO: 26 ) sequence of the tPA signal sequence fused to HIV-1_(LAI) gp120. The Trp₄₃₇ to Val CD4 binding mutation, the NarI and NotI restriction endonuclease sites used for cloning, and the predicted site of cleavage by signal peptidase between Arg₃₅ and Thr₃₆ are shown in bold.

FIG. 13

tPA-gp120_(JR-FL) -CD4(-).

Shown is the nucleotide (SEQ ID NO: 27) and deduced amino acid (SEQ ID NO: 28) sequence of the tPA signal sequence fused to HIV-1_(JR-FL) gp120. The Trp₄₂₄ to Val CD4 binding mutation, the NarI and NotI restriction endonuclease sites used for cloning and the predicted cleavage by signal peptidase between Arg₃₅ and Val₃₆ are shown in bold.

DETAILED DESCRIPTION OF THE INVENTION

The plasmids designated PPI4-tPA-gp120_(LAI) and PPI4-tPA-gp120_(JR-FL) were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 under ATCC Accession Nos. 75431 and 75432, respectively. The plasmids PPI4-tPA-gp120_(LAI) and PPI4-tPA-gp120^(JR-FL) were deposited with the ATCC on Mar. 12, 1993.

The subject invention provides a recombinant nucleic acid molecule which encodes a mutant HIV-1 gp120 envelope glycoprotein comprising a V3 loop deletion and a C4 domain.sub.(W→X) point mutation, wherein X is an amino acid residue other than tryptophan. In the preferred embodiment, X is a valine residue.

In one embodiment, the nucleic acid molecule is a DNA molecule. The DNA molecule may be a plasmid. In one embodiment, the plasmid comprises the sequence of the plasmid designated PPI4-tPA.

The V3 loop of HIV-1 gp120 envelope glycoprotein is shown in FIG. 1. The V3 loop is demarcated by cysteine residues at both its N- and C-termini. As used herein, a V3 loop deletion means a deletion of one or more amino acid residues between the terminal cysteine residues, with the proviso that there must be three or more amino acid residues situated between the two terminal cysteine residues in a V3 loop deletion. These three or more amino acid residues may either be residues originally present in the V3 loop, or exogenous residues. For example, as shown in the Experimental Details section infra, the pentapeptide TGAGH is situated between the two terminal cysteine residues. Variations in the size of the V3 loop deletion illustrated herein are tolerable without affecting the overall structure of the mutant HIV-1 gp120 envelope glycoprotein, as is well known to those skilled in the art.

As used herein, "C4 domain" means the HIV-1 gp120 envelope glycoprotein C4 domain having the following consensus sequence (SEQ ID NO: 1): ##STR1## wherein X₁ =T, I, V, K or R; X₂ =L, I or H; X₃ =P, Q, L or T; X₄ =R, K or G; X₅ =K or E; X₆ =Q or E; X₇ =F, I or V; X₈ =I, V or M; X₉ =N, R or K; X₁₀ =M, R, L or T; X₁₁ =Q, R or V; X₁₂ =E, K, G, R, V or A; X₁₃ =V, T, A or G; X₁₄ =G or E; X₁₅ =K, R, E, or Q; X₁₆ =M, V, I or L; X₁₇ =A, T or D; X₁₈ =P or L; X₁₉ =I or F; X₂₀ =S, R, G, K, N, A, E or Q; X₂₁ =G or R; X₂₂ =Q, L, P, N, K, V, T, E or I; X₂₃ =I, V or L; X₂₄ =R, K, S, N, G, I, T, E or I; X₂₅ =C or R; X₂₆ =S, L, I, T, P, E, V, K, D or N; X₂₇ =N, K or L; X₂₈ =I or V; X₂₉ =L, P or I; X₃₀ =L or I; X₃₁ =L or I; X₃₂ =T, A, I, V or E; X₃₃ =D or E; X₃₄ =G or V.

The C4 domain consensus sequence is based on existing C4 domain sequence information from various HIV-1 strains, and thus is not necessarily an exhaustive consensus sequence. The conserved tryptophan residue shown in bold after residue X₁₀ is the only conserved tryptophan residue in the C4 domain. As used herein, a C4 domain.sub.(W→X) point mutation is a mutation of the above-identified conserved C4 domain tryptophan residue to an amino acid residue other than tryptophan. For example, a C4 domain.sub.(W→V) point mutation is a mutation of the conserved C4 domain tryptophan residue to a valine residue.

In one embodiment, the C4 domain is an HIV-1_(LAI) gp120 envelope glycoprotein C4 domain. The sequence of the HIV-1_(LAI) gp120 C4 domain is: TLPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGG (SEQ ID NO: 2). The mutant HIV-1 gp120 envelope glycoprotein may be a mutant HIV-1_(LAI) gp120 envelope glycoprotein.

In another embodiment, the C4 domain is an HIV-1_(JR-FL) gp120 envelope glycoprotein C4 domain. The sequence of the HIV-1_(JR-FL) gp120 C4 domain is: TLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGG (SEQ ID NO: 3). The mutant HIV-1 gp120 envelope glycoprotein may be a mutant HIV-1_(JR-FL) gp120 envelope glycoprotein.

HIV-1_(LAI) is a laboratory-adapted strain that is tropic for phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) and immortalized human T-cell lines. In contrast, HIV-1_(JR-FL) was isolated from brain tissue taken at autopsy that was co-cultured with lectin-activated normal human PBLs. HIV-1_(JR-FL) is tropic for PHA-stimulated PBLs and blood-derived macrophages but will not replicate in transformed T-cell lines. Mutant HIV-1 gp120 envelope glycoproteins derived from a clinical isolate of HIV-1 such as JR-FL may possess new or different epitopes compared to the laboratory-adapted HIV-1 strains that are beneficial for successful vaccination. Although only the HIV-1_(LAI) and HIV-1_(JR-FL) strains are used herein to generate the mutant HIV-1 gp120 envelope glycoproteins of the subject invention, other HIV-1 strain could be substituted in their place as is well known to those skilled in the art.

The V1 and V2 variable regions of gp120 are unnecessary for CD4 binding (21). Therefore the mutant HIV-1 gp120 envelope glycoprotein of this invention can either include or exclude the V1 and V2 variable regions.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1 gp120 envelope glycoprotein comprising a V3 loop deletion and a C4 domain.sub.(Asp→X) point mutation, wherein the aspartate residue is between amino acid residues X₁₅ and X₁₆ in the C4 consensus sequence, and X is an amino acid residue other than aspartate or glutamate. In the preferred embodiment, X is an alanine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1 gp120 envelope glycoprotein comprising a V3 loop deletion and a C4 domain.sub.(Glu→X) point mutation, wherein the glutamate residue is between amino acid residues X₁₅ and X₁₆ in the C4 consensus sequence, and X is an amino acid residue other than aspartate or glutamate. In the preferred embodiment, X is an alanine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1_(LAI) gp120 envelope glycoprotein comprising a V3 loop deletion and a C3 domain.sub.(asp378→X) point mutation, wherein X is an amino acid residue other than aspartate or glutamate. In the preferred embodiment, X is a lysine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1_(JR-FL) gp120 envelope glycoprotein comprising a V3 loop deletion and a C3 domain.sub.(asp369→X) point mutation, wherein X is an amino acid residue other than aspartate or glutamate. In the preferred embodiment, X is a lysine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1_(LAI) gp120 envelope glycoprotein comprising a V3 loop deletion and a C3 domain.sub.(glu380→X) point mutation, wherein X is an amino acid residue other than glutamate. In the preferred embodiment, X is a glutamine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1_(JR-FL) gp120 envelope glycoprotein comprising a V3 loop deletion and a C3 domain.sub.(glu371→X) point mutation, wherein X is an amino acid residue other than glutamate. In the preferred embodiment, X is a glutamine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1_(LAI) gp120 envelope glycoprotein comprising a V3 loop deletion and a C2 domain.sub.(thr267→X) point mutation, wherein X is an amino acid residue other than threonine. In the preferred embodiment, X is an arginine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1_(JR-FL) gp120 envelope glycoprotein comprising a V3 loop deletion and a C2 domain(thr260→X) point mutation, wherein X is an amino acid residue other than threonine. In the preferred embodiment, X is an arginine residue.

The subject invention additionally provides a recombinant nucleic acid molecule which encodes a mutant HIV-1 gp120 envelope glycoprotein comprising (a) a V3 loop deletion, or (b) a one of the C2, C3 or C4 domain point mutations discussed supra.

The point mutations in the recombinant nucleic acid molecules described supra are selected based on their ability to reduce the affinity of the mutant gp120 glycoprotein encoded thereby for CD4. As used herein, the term "reduce the affinity" means to reduce the affinity by at least two-fold.

One skilled in the art would know how to make recombinant nucleic acid molecules which encode mutant HIV-1 gp120 envelope glycoproteins comprising a V3 loop deletion and the specific C2, C3 or C4 domain point mutations corresponding to those mutations exemplified in the HIV-1_(JR-FL) and HIV-1_(LAI) strains, supra. Furthermore, one skilled in the art would know how to use these recombinant nucleic acid molecules to obtain the proteins encoded thereby, and practice the therapeutic and prophylactic methods of using same, as described herein for the recombinant nucleic acid molecule which encodes a mutant HIV-1 gp120 envelope glycoprotein comprising a V3 loop deletion and a C4 domain.sub.(W→X) point mutation.

The subject invention also provides the mutant HIV-1 gp120 envelope glycoprotein encoded by the recombinant nucleic acid molecule of the subject invention.

In accordance with the invention, numerous vector systems for expression of the mutant HIV-1 gp120 envelope glycoprotein may be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), Semliki Forest virus or SV40 virus. Additionally, cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide resistance, (e.g., antibiotics) or resistance to heavy metals such as copper or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals. The cDNA expression vectors incorporating such elements include those described by Okayama (22).

The vectors used in the subject invention are designed to express high levels of mutant HIV-1 gp120 envelope glycoproteins in cultured eukaryotic cells as well as efficiently secrete these proteins into the culture medium. The targeting of the mutant HIV-1 gp120 envelope glycoproteins into the culture medium is accomplished by fusing in-frame to the mature N-terminus of the mutant HIV-1 gp120 envelope glycoprotein the tissue plasminogen activator (tPA) prepro-signal sequence.

The mutant HIV-1 gp120 envelope glycoprotein may be produced by a) transfecting a mammalian cell with an expression vector for producing mutant HIV-1 gp120 envelope glycoprotein; b) culturing the resulting transfected mammalian cell under conditions such that mutant HIV-1 gp120 envelope glycoprotein is produced; and c) recovering the mutant HIV-1 gp120 envelope glycoprotein so produced.

Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors may be transfected or introduced into an appropriate mammalian cell host. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniques. In the case of protoplast fusion, the cells are grown in media and screened for the appropriate activity. Expression of the gene encoding a mutant HIV-1 gp120 envelope glycoprotein results in production of the mutant glycoprotein.

Methods and conditions for culturing the resulting transfected cells and for recovering the mutant HIV-1 gp120 envelope glycoprotein so produced are well known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed.

In accordance with the claimed invention, the preferred host cells for expressing the mutant HIV-1 gp120 envelope glycoprotein of this invention are mammalian cell lines. Mammalian cell lines include, for example, monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line 293; baby hamster kidney cells (BHK); Chinese hamster ovary-cells-DHFR (CHO); Chinese hamster ovary-cells DHFR⁻ (DXB11); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); mouse cell line (C127); and myeloma cell lines.

Other eukaryotic expression systems utilizing non-mammalian vector/cell line combinations can be used to produce the mutant HIV-1 gp120 envelope glycoproteins. These include, but are not limited to, baculovirus vector/insect cell expression systems and yeast shuttle vector/yeast cell expression systems.

Methods and conditions for purifying mutant HIV-1 gp120 envelope glycoproteins from the culture media are provided in the invention, but it should be recognized that these procedures can be varied or optimized as is well known to those skilled in the art.

The subject invention further provides a vaccine which comprises a therapeutically effective amount of the mutant HIV-1 gp120 envelope glycoprotein of the subject invention, and an adjuvant.

A therapeutically effective amount of the mutant HIV-1 gp120 envelope glycoprotein may be determined according to methods well known to those skilled in the art.

As used herein, adjuvants include, but are not limited to, alum, Freund's incomplete adjuvant (FIA), Saponin, Quil A, Monophosphoryl lipid A (MPL), and nonionic block copolymers (SAF) such as L-121 (Pluronic; SYNTEX SAF (nonionic block copolymers)). In the preferred embodiment, the adjuvant is alum, especially in the form of a thixotropic, viscous, and homogeneous aluminum hydroxide gel. The vaccine of the subject invention may be administered as an oil in water emulsion. Methods of combining adjuvants with antigens are well known to those skilled in the art.

The subject invention further provides a method of treating an HIV-1-infected subject, which comprises immunizing the HIV-1-infected subject with the vaccine of the subject invention, thereby treating the HIV-1-infected subject.

As used herein, treating an HIV-1-infected subject with the vaccine of the subject invention means reducing in the subject either the population of HIV-1 or HIV-1-infected cells, or ameliorating the progression of an HIV-1-related disorder in the subject.

As used herein, an "HIV-infected subject" means an individual having at least one of his own cells invaded by HIV-1.

As used herein, "immunizing" means administering a primary dose of the vaccine to a subject, followed after a suitable period of time by one or more subsequent administrations of the vaccine, so as to generate in the subject an immune response against the CD4-binding region of the mutant HIV-1 gp120 envelope glycoprotein in the vaccine. A suitable period of time between administrations of the vaccine may readily be determined by one skilled in the art, and is usually in the order of several weeks to months.

In the preferred embodiment, the dose of vaccine administered is an amount sufficient to deliver to the subject between 10 ug and 1 mg of the mutant HIV-1 gp120 envelope glycoprotein.

The subject invention further provides a vaccine which comprises a prophylactically effective amount of the mutant HIV-1 gp120 envelope glycoprotein of the subject invention, and an adjuvant.

A prophylactically effective amount of the mutant HIV-1 gp120 envelope glycoprotein may be determined according to methods well known to those skilled in the art.

The subject invention further provides a method of reducing the likelihood of an HIV-1-exposed subject's becoming infected with HIV-1, which comprises immunizing the HIV-1-exposed subject with the vaccine of the subject invention, thereby reducing the likelihood of the HIV-1-exposed subject's becoming infected with HIV-1.

As used herein, the subject's becoming infected with HIV-1 means the invasion of the subject's own cells by HIV-1.

As used herein, reducing the likelihood of a subject's becoming infected with HIV-1 means reducing the likelihood of the subject's becoming infected with HIV-1 by at least two-fold. For example, if a subject has a 1% chance of becoming infected with HIV-1, a two-fold reduction in the likelihood of the subject's becoming infected with HIV-1 would result in the subject's having a 0.5% chance of becoming infected with HIV-1. In the preferred embodiment of this invention, reducing the likelihood of the subject's becoming infected with HIV-1 means reducing the likelihood of the subject's becoming infected with HIV-1 by at least ten-fold.

As used herein, an HIV-1-exposed subject is a subject who has HIV-1 present in his body, but has not yet become HIV-1-infected.

The subject invention further provides a method of reducing the likelihood of a non-HIV-1-exposed subject's becoming infected with HIV-1, which comprises immunizing the non-HIV-1-exposed subject with the vaccine of the subject invention, thereby reducing the likelihood of the non-HIV-1-exposed subject's becoming infected with HIV-1.

As used herein, a non-HIV-1-exposed subject is a subject who does not have HIV-1 present in his body.

The subject invention further provides a method of obtaining partially purified antibodies which specifically bind to the CD4-binding domain of HIV-1 gp120 envelope glycoprotein, which method comprises (a) immunizing a non-HIV-1-exposed subject with the vaccine of the subject invention, (b) recovering from the immunized subject serum comprising said antibodies, and (c) partially purifying said antibodies, thereby obtaining partially purified antibodies which specifically bind to the CD4-binding domain of HIV-1 gp120 envelope glycoprotein. In the preferred embodiment, the subject is a human.

As used herein, partially purified antibodies means a composition which comprises antibodies which specifically bind to the CD4-binding domain of HIV-1 gp120 envelope glycoprotein, and consists of fewer protein impurities than does the serum from which the anti-CD4-binding domain antibodies are derived. A protein impurity means a protein other than the anti-CD4-binding domain antibodies. For example, the partially purified antibodies might be an IgG preparation.

Methods of recovering serum from a subject are well known to those skilled in the art. Methods of partially purifying antibodies are also well known to those skilled in the art, and include, by way of example, filtration, ion exchange chromatography, and precipitation.

In one embodiment, the partially purified antibodies comprise an immune globulin (IG) preparation. IG can be purified from serum by a two-step process. Initially, serum is fractionated by the cold ethanol method of Cohn, et al. (29). Cohn Fraction II has as its main protein component IgG immunoglobulin present as monomers, dimers and aggregates. Fraction II is then purified to produce IVIG (immune globulin intravenous) using a variety of purification methods which include, for example, ion exchange, DEAE chromatography, acid pH 4.25 diafiltration, PEG precipitation or Pepsin treatment. The final product is stabilized (e.g., glucose+NaCl) and the final IgG concentration is fixed at between about 3% and about 6%.

The subject invention further provides the partially purified antibodies produced by the method of the subject invention.

The subject invention further provides a pharmaceutical composition, which comprises a therapeutically effective amount of the partially purified antibodies of the subject invention, and a pharmaceutically acceptable carrier.

A therapeutically effective amount of the partially purified antibodies of the subject invention may be determined according to methods well known to those skilled in the art.

Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

The subject invention further provides a method of treating an HIV-1-infected subject, which comprises administering to the subject a dose of the pharmaceutical composition of the subject invention effective to reduce the population of HIV-1-infected cells in the HIV-1-infected subject, thereby treating the HIV-1-infected subject.

As used herein, administering may be effected or performed using any of the various methods known to those skilled in the art. The administering may comprise administering intravenously. The administering may also comprise administering intramuscularly. The administering may further comprise administering subcutaneously.

The dose of the pharmaceutical composition of the subject invention effective to reduce the population of HIV-1-infected cells in the HIV-1-infected subject may be readily determined using methods well known to those skilled in the art. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 10 mg/kg and 150 mg/kg of protein if administered intramuscularly. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 100 mg/kg and 2 g/kg of protein if administered intravenously.

The subject invention further provides a method of treating an HIV-1-infected subject, which comprises administering to the subject a dose of the pharmaceutical composition of the subject invention effective to reduce the population of HIV-1 in the HIV-1-infected subject, thereby treating the HIV-1-infected subject.

The dose of the pharmaceutical composition of the subject invention effective to reduce the population of HIV-1 in the HIV-1-infected subject may be readily determined using methods well known to those skilled in the art. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 10 mg/kg and 150 mg/kg of protein if administered intramuscularly. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 100 mg/kg and 2 g/kg of protein if administered intravenously.

The subject invention further provides a composition which comprises a prophylactically effective amount of the partially purified antibodies of the subject invention, and a pharmaceutically acceptable carrier.

A prophylactically effective amount of the partially purified antibodies of the subject invention may be determined according to methods well known to those skilled in the art.

The subject invention further provides a method of reducing the likelihood of an HIV-1-exposed subject's becoming infected with HIV-1, which comprises administering to the HIV-1-exposed subject a dose of the composition of the subject invention effective to reduce the population of HIV-1 in the HIV-1-exposed subject, thereby reducing the likelihood of the subject's becoming infected with HIV-1.

In one embodiment, the subject is a medical practitioner. The medical practitioner may be a medical practitioner exposed to an HIV-1-containing bodily fluid. As used herein, the term "medical practitioner" includes, but is in no way limited to, doctors, dentists, surgeons, nurses, medical laboratory assistants, and students in health care programs.

In another embodiment, the subject is a newborn infant. The newborn infant may be a newborn infant born to an HIV-1-infected mother.

The dose of the composition of the subject invention effective to reduce the population of HIV-1 in the HIV-1-exposed subject may be readily determined using methods well known to those skilled in the art. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 10 mg/kg and 150 mg/kg of protein if administered intramuscularly. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 100 mg/kg and 2 g/kg of protein if administered intravenously.

The vaccines and pharmaceutical compositions of the subject invention may also ameliorate the progression of an HIV-1-related disorder in a subject to whom the vaccines or pharmaceutical compositions were administered while the subject was either non-HIV-1-exposed or HIV-1-exposed, but not yet HIV-1-infected.

Finally, the subject invention provides a method of reducing the likelihood of a non-HIV-1-exposed subject's becoming infected with HIV-1 as a result of exposure thereto during an incident wherein there is an increased risk of exposure to HIV-1, which comprises administering to the subject immediately prior to the incident a dose of the composition of the subject invention effective to reduce the population of HIV-1 to which the subject is exposed during the incident, thereby reducing the likelihood of the subject's becoming infected with HIV-1. In one embodiment, the subject is a medical practitioner.

An incident wherein there is an increased risk of exposure to HIV-1 includes, for example, receiving a blood transfusion, sexual contact with an HIV-1-infected individual, and performing a HIV-1-containing bodily fluid-exposing medical procedure.

As used herein, "immediately prior to the incident" means within one month of the incident. In the preferred embodiment, "immediately prior to the incident" means within one day of the incident.

The dose of the composition of the subject invention effective to reduce the population of HIV-1 to which the subject is exposed during the incident may be readily determined using methods well known to those skilled in the art. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 10 mg/kg and 150 mg/kg of protein if administered intramuscularly. In the preferred embodiment, the dose is sufficient to deliver to the subject between about 100 mg/kg and 2 g/kg of protein if administered intravenously.

One embodiment of this invention is a method of substantially reducing the likelihood of a non-infected medical practitioner's becoming infected with HIV-1 during a bodily fluid-exposing medical procedure involving a patient, which comprises administering to the patient during a suitable time period an amount of the composition of the subject invention effective to substantially reduce the likelihood of the non-infected medical practitioner's becoming infected with HIV-1 by virtue of contact with the patient's bodily fluid during the medical procedure.

As used herein, a bodily fluid is any fluid which is present in the human body and is capable of containing infectious HIV-1 in an HIV-1-infected patient. Bodily fluids include, but are not limited to, saliva, cerebrospinal fluid, tears, vaginal secretions, urine, alveolar fluid, synovial fluid and pleural fluid.

Another embodiment of this invention is a method of substantially reducing the likelihood of a non-HIV-1-infected newborn infant's becoming infected with HIV-1 prior to or during birth from an HIV-1-infected mother, which comprises administering to the mother prior to birth an amount of the composition of the subject invention effective to substantially reduce the likelihood of the non-HIV-1-infected newborn infant's becoming infected with HIV-1 by virtue of contact with the patient's bodily fluid.

In order to facilitate an understanding of the Experimental Details section which follows, certain frequently occurring methods and/or terms are best described in Maniatis et al. (23).

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS Nomenclature

As used herein, V3(-) indicates a V3 loop deletion from HIV-1 gp120 envelope glycoprotein. As used herein, CD4(-) indicates a point mutation in the C4 domain of HIV-1 gp120 envelope glycoprotein which mutation inhibits CD4 binding to the mutant HIV-1 gp120 envelope glycoprotein. The structure of HIV-1 gp120 envelope glycoprotein is shown in FIG. 1.

Materials and Methods

1. Construction of PPI4-tPA-gp120_(LAI) expression vector.

An expression vector was constructed that consisted of the cytomegalovirus major immediate early (CMV MIE) promoter/enhancer linked to the HIV-1_(LAI) env gene, which gene had its signal sequence replaced by the tPA signal sequence. The CMV MIE promoter/enhancer sequences were derived from pSVCC1 (24) consisting of 1580 base pairs of contiguous DNA that is immediately 5' to the initiator ATG. In sequential order, the functional domains of the CMV promoter are: the promoter/enhancer region; a transcriptional initiator site; exon A (a non-coding exon); intron A; and 17 nucleotides of exon B (non-coding sequences). The viral promoter sequences were ligated to a gene construct consisting of the nucleotide sequences encoding amino acids -35 to -1 of human tPA (25) fused in-frame to HIV-1_(LAI) env amino acids 31 through 515, ending with a TGA stop codon. The construction was performed in two parts. The majority of the CMV promoter could be isolated as a 1560 bp Hinc II/Pst I fragment which was ligated to a Pst I/Not I 1590 bp DNA fragment that contained the remainder of the CMV promoter, the initiator ATG, the tPA signal sequence and the mature HIV-1_(LAI) env protein coding sequence.

The latter fragment was assembled using the polymerase chain reaction as follows. Primer 1 (GATCCTGCAGTCACCGTCCTTGACACGATGGATGCAATGAAGAGA (SEQ ID NO 4)) and primer 2 (AAGTCTTCTCCTCGGTCTTGTCTTTTTAACACCCAG (SEQ ID NO: 5) were used to amplify the nucleic acid sequences encoding the tPA signal sequence amino acids -35 to -1 from plasmid pMAM neo-s (Clonetech), thus producing a 150 bp fragment. A second 1440 bp DNA fragment was amplified using primer 3 (TTCAGAAGAGGAGCCAGAACAGAAAAATTGTGGGTC (SEQ ID NO: 6)), primer 4 (GGAAAAAAGCGGCCGCTCATTTTTCTCTCTGCACCACTC), and pENV (26) as a template. The PCR fragments were pooled, desalted, and excess primer removed by ultrafiltration through a centricon-100 unit (Amicon). An aliquot of the pooled material was then subjected to a second round of amplification in the presence of primers 1 and 4 to produce a 1590 bp fragment, which was then digested with Pst I and Not I. The CMV promoter fragment and the HIV-1LAI env fragment were then ligated together, and the entire transcription unit subcloned into PPI4, which is a eukaryotic shuttle vector that contains an ampicillin resistance gene, an SV40 origin of replication and a DHFR gene whose transcription is driven by the β-globin promoter. The final construct, PPI4-tPA-gp120_(LAI), is shown in FIG. 2.

The expression vector is then used as the prototype vector for the expression of gp120 proteins that are derived from other HIV-1 strains or mutated as described in the methods section. The vector was constructed so that unique Nar I and Not I sites flank the gp120 sequence, thus facilitating the removal of the gp120 gene cassette and the subsequent insertion of other gene cassettes (FIG. 2).

2. Expression of HIV-1_(LAI) gp120 in mammalian cells.

a. Transient expression.

CosM5 cells grown in DMEM containing 10% fetal calf serum were split to 75% confluence. On the following day, the cells were transfected for 16-20 hours with 10 micrograms of CsCl-purified PPI4-tPA-gp120_(LAI) DNA by the standard CaPO₄ (5) precipitation technique. After transfection, fresh medium was added to the cells. Analysis of the products synthesized 96-120 hours post-transfection was performed by radiolabelling the transfectants with ³⁵ S-cysteine for 12-18 hours, followed by precipitation of media using a CD4-immunoglobulin-Protein A-Sepharose complex, followed by SDS-PAGE under reducing conditions (FIG. 4).

b. Stable expression.

Dhfr- Chinese hamster ovary cells (CHO) were transfected with 20 micrograms of CsCl-purified DNA. Approximately 3-5 days post-transfection, cells were placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum). Approximately 10-15 days post-selection, individual cell clones were picked. Media was analyzed for gp120 expression by radiolabelling the cells with ³⁵ S-cysteine for 12-18 hours, followed by precipitation of media using a CD4-immunoglobulin-Protein A-Sepharose complex, followed in turn by SDS-PAGE under reducing conditions (FIG. 6). The levels of gp120 in the media of these clones were also quantitated (FIG. 5) by ELISA performed as follows. The method involves coating 96-well plates overnight with sheep polyclonal IgG against the highly conserved C-terminus of gp120 (D7234, Aalto Bioreagents). After washing, dilutions of a standard gp120 preparation in cell growth medium, or supernatant from the stably-transfected cells, were incubated for 1 hour. The plates were washed again, and incubated for one hour with a horseradish peroxidase-conjugated anti-gp120 monoclonal antibody (9204, DuPont). Following a final wash, the peroxidase substrate OPD (DuPont) was added and the amount of gp120 determined by comparing absorbance of unknowns with a standard curve. Standards were prepared from purified gp120 made in CHO cells, a small quantity of which was obtained from Celltech Ltd. Clones expressing the highest levels were subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines were thus generated which secrete at least 1 microgram/milliliter of HIV-1_(LAI) gp120.

3. Construction of PPI4-tPA-gp120_(JR-FL).

a. The HIV-1_(LAI) gp120 env nucleotide sequence in PPI4-tPA-gp120_(LAI) was replaced by the nucleotide sequence encoding the mature gp120_(JR-FL) protein. Using the polymerase chain reaction, the JR-FL sequences were amplified from pUC112-1 (27) using primer 5 (GATCGGCGCCAGAGTAGAAAAGTTGTGGGTCAC (SEQ ID NO: 8)) and primer 4. The PCR fragment was digested with the restriction endonucleases Nar I and Not I, and the fragment subcloned in between the Nar I and Not I sites in PPI4-tPA-gp120_(LAI) to generate PPI4-tPA-gp120_(JR-FL) (FIG. 7).

b. Transient expression.

CosM5 cells grown in DMEM containing 10% fetal calf serum were split to 75% confluence. On the following day, the cells were transfected for 16-20 hours with 10 micrograms of CsCl-purified PPI4-tPA-gp120_(JR-FL) DNA by the standard CaPO₄ (5) precipitation technique. After transfection, fresh medium was added to the cells. Analysis of the products synthesized 96-120 hours post-transfection was performed by radiolabelling the transfectants with ³⁵ S-cysteine for 12-18 hours, followed by precipitation of media using a CD4-immunoglobulin-Protein A-Sepharose complex, followed by SDS-PAGE under reducing conditions (FIG. 4).

4. Construction of PPI4-tPA-gp120_(LAI) -V3(-).

The V3 loop in tPA-gp120_(LAI) consists of amino acids CYs₃₀₆ through Cys₃₃₃. In the V3(-) mutant, the amino acids in between these cysteines are replaced by the pentapeptide sequence Thr-Gly-Ala-Gly-His. Using the Transformer Site- Directed Mutagenesis Kit (Clonetech), the V3 loop sequence in PPI4-tPA-gp120_(LAI) is altered using the mutagenic primer 6 (CTGTAGAAATTAATTGTACAGGTGCTGGACATTGTAACATTAGTAGAGC (SEQ ID NO: 9)) and primer 7 (CTCGAGCATGCATTCGAAGCTCGCTGATC (SEQ ID NO: 10)) as a selection primer. Primer 7 changes a unique Xba I site in the backbone of the parent PPI4 plasmid into a unique BstB I site. Briefly, the mutagenesis method requires incubating of the parent plasmid with the mutagenic primer and the selection primer, denaturing at 100° C. for 3 minutes and then chilling on ice. In the presence of buffered deoxynucleo-tide triphosphates and T4 DNA polymerase, the primers are allowed to initiate the polymerization of one strand of plasmid DNA. T4 DNA ligase is used to seal the newly synthesized DNA strand to form a covalently closed circle. Hybrid plasmids are then transformed into a MutS strain of E. coli that is deficient in mismatch repair. After allowing for the growth of transformed cells, DNA is purified from the cells and digested with the selection restriction endonuclease, in this case Xba I. Parental plasmids are cleaved by Xba I while the mutant plasmid remains resistant to cleavage by virtue of the Xba I to BstB I conversion. Digested DNA is then used to transform E. coli, and colonies harboring the mutant plasmid are picked. Multiple mutagenic primers can be used in a single round of mutagenesis. The amino acid sequence of the modified protein is shown in FIG. 8.

5. Construction of PPI4-tPA-gp120_(JR-FL) -V3(-).

The V3 loop in tPA-gp120_(JR-FL) consists of amino acids Cys₂₉₃ through CyS₃₂₇. In the V3(-) mutant, the amino acids in between these cysteines are replaced by the pentapeptide sequence Thr-Gly-Ala-Gly-His. Using the Transformer Site-Directed Mutagenesis Kit (Clonetech), the V3 loop sequence in PPI4-tPA-gp120_(JR-FL) is altered using the mutagenic primer 6 (CTGTAGAAATTAATTGTACAGGTGCTGGACATTGTAACATTAGTAGAGC (SEQ ID NO: 9)) and primer 7 as a selection primer. The amino acid sequence of the modified protein is shown in FIG. 9.

6. Construction of PPI4-tPA-gp120_(LAI) -CD4(-).

Using the Transformer Site-Directed Mutagenesis Kit (Clonetech), the selection primer 7, and the mutagenic primer 8 (CAATTTATAAACATGGTGCAGGAAGTAGG (SEQ ID NO: 11), Trp₄₃₇ of tPA-gp120_(LAI), which is in an equivalent position to the tryptophan residue in the HXBc2 strain of HIV-1, is mutated to a Val in the expression vector PPI4-tPA-gp120_(LAI) to generate PPI4-tPA-gp120_(LAI) -CD4(-). The sequence for gp120_(LAI) -CD4(-) is shown in FIG. 12.

7. Construction of PPI4-tPA-gp120_(JR-FL) -CD4 (-).

In a fashion similar to that described above, Trp₄₂₄ of tPA-gp120_(JR-FL) is mutated to a Val in the expression vector PPI4-tPA-gp120_(JR-FL) using the selection primer 7 and the mutagenic primer 9 (CAAATTATAAACATGGTGCAGGAAGTAGG (SEQ ID NO: 12)) to generate PPI4-tPA-gp120_(JR-FL) -CD4(-). The sequence for gp120_(JR-FL) -CD4(-) is shown in FIG. 13.

8. Construction of PPI4-tPA-gp120_(LAI) -V3(-)-CD4(-).

The tPA-gp120_(LAI) double mutant, V3(-)-CD4(-), is constructed by including the mutagenic primers 6 and 8, and the selection primer 7 simultaneously in the reaction tube with PPI4-tPA-gp120_(LAI) as the DNA template. The final construct is named PPI4-tPA-gp120_(LAI) -V3(-)-CD4(-), and its sequence is shown in FIG. 10.

9. Construction of PPI4-tPA-gp120_(JR-FL) -V3(-)-CD4(-).

The tPA-gp120_(JR-FL) double mutant, V3(-)-CD4(-), is constructed by including the mutagenic primers 6 and 9, and the selection primer 7 simultaneously in the reaction tube with PPI4-tPA-gp120_(JR-FL) as the DNA template. The final construct is named PPI4-tPA-gp120_(JR-FL) -V3(-)-CD4(-), and its sequence is shown in FIG. 11.

10. Expression of mutant HIV-1 gp120 in mammalian cells.

a. Transient expression.

CosM5 cells grown in DMEM containing 10% fetal calf serum are split to 75% confluence. On the next day, the cells are transfected for 16-20 hours with 10 micrograms of CsCl-purified mutant HIV-1 DNA by the standard CaPO₄ (5) precipitation technique. After transfection, fresh medium is added to the cells. Analysis of the products synthesized 96-120 hours post-transfection is performed by radiolabelling the transfectants with ³⁵ S-cysteine for 12-18 hours, followed by precipitation of media using a sheep polyclonal IgG against the highly conserved C-terminus of gp120.

b. Stable expression.

Dhfr⁻ Chinese hamster ovary cells (CHO) are transfected with 20 micrograms of CsCl-purified DNA encoding the native or mutant HIV-1 gp120 glycoproteins. Approximately 3-5 days post-transfection, cells are placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum). Approximately 10-15 days post-selection, individual cell clones are picked. Media is analyzed for gp120 expression by radiolabelling the cells with 35S-cysteine for 12-18 hours, followed by quantitative immunoprecipitation of media using a sheep polyclonal IgG against the highly conserved C-terminus of gp120, followed in turn by SDS-PAGE under reducing conditions. Alternatively, one can quantitate the level of gp120 by ELISA performed as follows. The method involves coating 96-well plates overnight with sheep polyclonal IgG against the highly conserved C-terminus of gp120 (D7234, Aalto Bioreagents). After washing, dilutions of a standard gp120 preparation in cell growth medium, or supernatant from the stably-transfected cells, are incubated for 1 hour. The plates are washed again, and incubated for one hour with a human MoAb (F105, AIDS Research & Reference Reagent Program, No. 857). The plates are washed again, and incubated again for 1 hour with a horseradish-peroxidase-conjugated goat anti-human IgG (Cappel). Following a final wash, the peroxidase substrate OPD (DuPont) is added and the amount of gp120 determined by comparing absorbance of unknowns with a standard curve. Standards are prepared from purified gp120 made in CHO cells, a small quantity of which is obtained from Celltech Ltd. Clones expressing the highest levels are subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines are thus generated which secrete at least 1 microgram/milliliter of mutant HIV-1 gp120.

11. Purification of HIV-1 gp120 proteins.

A one-step immunoaffinity procedure is used to purify the recombinant gp120 molecules described. Briefly, culture supernatant is collected and clarified by centrifugation. An immunoaffinity column consisting of a matrix coupled to a sheep polyclonal anti-gp120 IgG (D7234, Aalto Bioreagents) directed against the highly conserved C-terminal end (APTKAKRRVVQREKR (SEQ ID NO: 29) of gp120 is used to specifically adsorb gp120 from the cell culture media. This antisera recognizes native gp120, the V3 loop deletion mutants, and the CD4(-) mutants since the C-terminal ends of these molecules remain unaltered. The bound gp120 is then eluted with 2M MgCl₂, concentrated by Amicon filtration, and dialyzed into 10 mM HEPES, pH 7.0. The purity of the proteins is determined by SDS-PAGE and silver staining.

12. Characterization of recombinant HIV-1 gp120 proteins.

The purified glycoproteins are subjected to extensive biochemical and immunologic characterization. The integrity of the proteins is monitored by SDS-PAGE and silver staining under reducing and non-reducing conditions. The glycoproteins are deglycosylated by treatment with the enzyme N-glycosidase F which cleaves N-linked oligo-saccharides, and are assayed by SDS-PAGE and silver staining to monitor molecular weight shifts. The purified glycoproteins are also tested for reactivity with several well characterized anti-gp120 monoclonal antibodies that recognize both linear and discontinuous epitopes. The binding affinity to sCD4 is estimated using an ELISA assay.

13. A protocol for inoculation of animals with the mutant HIV-1 gp120 envelope glycoproteins.

Alum is used as an adjuvant during the inoculation series. The inoculum is prepared by dissolving the mutant HIV-1 gp120 envelope glycoprotein antigen in physiologic saline at a final antigen concentration of 100 ug/ml. Preformed alum (aluminum hydroxide gel) is added to the solution to a final level of 500 ug/ml aluminum. The antigen is allowed to adsorb onto the alum gel for two hours at room temperature. Following adsorption, the gel with the antigen is washed twice with physiologic saline and resuspended in the saline to a protein concentration of 100 ug/ml.

Monkeys and/or Guinea Pigs are individually inoculated with four 100 ug doses of the mutant HIV-1 gp120 envelope glycoprotein antigen adsorbed onto alum. Each dose is injected intramuscularly. The doses are delivered one or five months apart (week 0, 4, 8 and 28). The animals are bled at intervals of two or four weeks. Serum samples are prepared from each bleed to assay for the development of specific antibodies as described in the subsequent sections.

14. Analysis of sera for anti-mutant HIV-1 gp120 envelope glycoprotein IgG antibodies.

Each serum sample is analyzed by ELISA. Polystyrene microtiter plates are coated with 0.5 ug per well of pure mutant HIV-1 gp120 envelope glycoprotein in phosphate-buffered physiological saline (PBS) at 4° C. Each well is then washed with PBS containing 0.5% TWEEN-20 (PBS-TW). Test serum, diluted serially in PBS-TW, is added to the mutant HIV-1 gp120 envelope glycoprotein-containing wells and allowed to react with the adsorbed mutant HIV-1 gp120 envelope glycoprotein for one hour at 37° C. The wells are then washed extensively in PBS-TW. Each well then receives 0.1% p-nitrophenyl phosphate in 10% diethanolamine, pH 9.8, containing 0.5 mM MgCl₂ ·6H₂ O. The ensuing reaction is allowed to proceed at room temperature for 30 minutes, at which time it is terminated by the addition of 3.0N NaOH.

The greater the interaction of antibodies in the test serum with the mutant HIV-1 gp120 envelope glycoprotein, the greater is the amount of alkaline phosphatase bound onto the well. The phosphatase enzyme mediates the breakdown of p-nitrophenyl phosphate into a molecular substance which absorbs light at a wavelength of 405 nm. Hence, there exists a direct relationship between the absorbance at 405 nm of light at the end of the ELISA reaction and the amount of mutant HIV-1 gp120 envelope glycoprotein-bound antibody. All animals inoculated with mutant HIV-1 gp120 envelope glycoprotein whose serum reacts specifically with the mutant HIV-1 gp120 envelope glycoprotein in the ELISA have a positive antibody response against mutant HIV-1 gp120 envelope glycoprotein.

15. Analysis of sera for activity which specifically neutralizes HIV-1 infectivity.

Virus-neutralizing activity is determined with an assay based on the use of multiplicity curves in which the ratio of infectious virus surviving antibody treatment (V_(n)) is compared to infectious virus in uninhibited cultures (V_(o)) at various dilutions of antisera. The neutralization titer of the sera is then interpolated as that sera dilution which yields one log reduction in infectious titer (i.e., V_(n) /V_(o) =0.1). Briefly, 4-fold dilutions of virus (laboratory-adapted and primary isolates) are prepared to yield infectious doses of 0.1 to 100 TCID₅₀ (Tissue Culture Infection Dose) in 20 ul. Serial 3-fold dilutions of sera are also prepared and 20 ul of each serum dilution are incubated with each dilution of virus in duplicate for 60 minutes at room temperature in a 96-well microtiter plate. 20 ul of AA5 cells (PHA stimulated PBMCs for primary HIV-1 isolates) are then added to the serum/virus mixtures. Cells are cultured for 7 days by the addition of fresh medium every other day. On the seventh day, supernatant from each well is removed and tested for the presence of reverse transcriptase (RT). Infection in each well is then scored as either positive or negative based on the RT counts, and the infectious dose of virus in each treatment group is calculated using the Reed and Muench (28) formula. The neutralization titers represent the reciprocal serum dilution required to reduced infectious dose of virus by one log. The above culture time is for the prototypic HIV-1_(LAI) isolate tested on the AA5 cell line. In the case of primary isolates, the termination date is usually 11-14 days. Culture conditions for PBMCs is not as demanding since doubling time is restricted. In the case of PBMCs, one day PHA stimulations are used at a final concentration of 1.5×10⁶ /ml on day 0. Half that number of fresh PBMCs are then added again on days 4 and 8. This multiple addition of PBMCs is meant to amplify virus output upon successful infection so that the readout RT signal is strong. Again, the final readout titer for the primary isolate/PBMC is the reciprocal serum dilution which reduces infectious titer by one log.

16. Passive hyperimmune therapy.

Non-HIV-1-infected humans are immunized with the mutant HIV-1 gp120 envelope glycoprotein antigens according to a protocol similar to that described above in section 12. For passive hyperimmune therapy in HIV-1-infected individuals, blood plasma is taken from mutant HIV-1 gp120 envelope glycoprotein immunized, non-HIV-1-infected human donors whose plasma has high levels of neutralizing antibodies. The plasma is pooled from several donors, purified to remove nonimmunoglobulin proteins and is then sterilized to kill any other viruses or pathogens. The treated plasma is then injected into individuals infected with HIV-1, with repeated injections every week, every two weeks, or every month.

Results

Eukaryotic expression vectors designed to express high levels of HIV-1_(LAI) gp120 and HIV-1_(JR-FL) gp120 were constructed. The CMV MIE promoter/enhancer was used to drive the transcription of a gene fusion consisting of the human tPA signal sequence fused to mature gp120 (FIGS. 2 and 7). The complete sequence of the transcription unit from the Hinc II site of the CMV promoter/enhancer to the Not I site just 3' from the stop codon in gp120 is shown in FIG. 3. This vector was used to transfect COSM5 cells in a transient assay. The transfected cells were labeled with ³⁵ S-cysteine and the media immunoprecipitated with a CD4-immunoglobulin-Protein A-Sepharose complex. The precipitated products were analyzed using a reducing 10% SDS-PAGE gel and autoradiography (FIG. 4). A 120 kD band was detected when PPI4-tPA-gp120_(LAI) was used to transfect COS cells (lane 3). A band migrating with a slightly lower molecular mass was detected when PPI4-tPA-gp120_(JR-FL) was used to transfect COS cells (lane 4). No radiolabeled products were detected in the mock infected cells. Using a sheep polyclonal antibody directed against the highly conserved C-terminal end of HIV-1 gp120 in an ELISA assay, the level of expression of HIV-1 gp120 was determined to be 2350 ng/ml.

The PPI4-tPA-gp120_(LAI) vector was then used to stably transfect the dhfr⁻ CHO cell line DXB11. Two days post-transfection, the cells were plated at low density in nucleoside-free medium. Eight days post-transfection, surviving clones were isolated and expanded. Individual primary transfectants were tested for gp120 expression using the ELISA method described in the methods section. Several primary CHO transfectants expressed significant quantities (10-120 ng/ml) of gp120 (FIG. 5). Three of the highest expressing clones were then subjected to increasing concentrations of methotrexate in order to amplify, in tandem, the copy number of the dhfr and gp120 genes. Cell lines were established that express high levels of gp120 with rates of secretion greater than 1 mg/liter. These were then used to purify gp120 to homogeneity.

Discussion

The advantage of using the mutant HIV-1 gp120 envelope glycoproteins as immunogens is that these proteins will not elicit an immune response against the V3 loop, a highly immunodominant epitope on gp120. This is significant because the V3 loop may skew the humoral immune response away from discontinuous epitopes in the CD4-binding site. Mutant HIV-1 gp120 envelope glycoproteins having partial and total V3 loop deletions have been made (30). Deletion of the V3 loop therefore exposes the CD4-binding site to the immune system, allowing the immune system to mount a response against this critical region (18). Another advantage of using the mutant HIV-1 gp120 envelope glycoprotein as an immunogen is that it has significantly reduced affinity for cell surface CD4. An efficient humoral immune response depends on the binding of antigen to B cell surface immunoglobulin. The presence of the high-affinity CD4 receptor on large numbers of cells in the body may significantly diminish the ability of native gp120 to induce an effective humoral immune response. The rationale of mutating gp120 at the CD4 binding site is to redirect the mutant HIV-1 gp120 envelope glycoprotein away from cell surface CD4 toward immunoglobulin-bearing B cells, thereby allowing the immune system to mount a response against, inter alia, the CD4-binding site.

References

1. Klatzmann, D. R., et. al. (1990) Immunodeficiency Reviews 2, 43-66.

2. Lasky, L. A., et. al. (1987) Cell 50, 975-985.

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    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 29     (2) INFORMATION FOR SEQ ID NO:1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 45 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     XaaXaaXaaCysXaaIleXaaXaaXaaXaaXaaXaaTrpXaaXaaXaa     151015     XaaXaaAlaXaaTyrXaaXaaProXaaXaaXaaXaaXaaXaaXaaXaa     202530     SerXaaXaaThrGlyXaaXaaXaaXaaArgXaaGlyXaa     354045     (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 45 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     ThrLeuProCysArgIleLysGlnPheIleAsnMetTrpGlnGluVal     151015     GlyLysAlaMetTyrAlaProProIleSerGlyGlnIleArgCysSer     202530     SerAsnIleThrGlyLeuLeuLeuThrArgAspGlyGly     354045     (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 45 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     ThrLeuProCysArgIleLysGlnIleIleAsnMetTrpGlnGluVal     151015     GlyLysAlaMetTyrAlaProProIleArgGlyGlnIleArgCysSer     202530     SerAsnIleThrGlyLeuLeuLeuThrArgAspGlyGly     354045     (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 45 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     GATCCTGCAGTCACCGTCCTTGACACGATGGATGCAATGAAGAGA45     (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 36 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     AAGTCTTCTCCTCGGTCTTGTCTTTTTAACACCCAG36     (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 36 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     TTCAGAAGAGGAGCCAGAACAGAAAAATTGTGGGTC36     (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 39 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     GGAAAAAAGCGGCCGCTCATTTTTCTCTCTGCACCACTC39     (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 33 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     GATCGGCGCCAGAGTAGAAAAGTTGTGGGTCAC33     (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 49 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     CTGTAGAAATTAATTGTACAGGTGCTGGACATTGTAACATTAGTAGAGC49     (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 29 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     CTCGAGCATGCATTCGAAGCTCGCTGATC29     (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 29 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     CAATTTATAAACATGGTGCAGGAAGTAGG29     (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 29 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     CAAATTATAAACATGGTGCAGGAAGTAGG29     (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 3125 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1555..3115     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     TTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAG60     CCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC120     CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG180     GACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACA240     TCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC300     CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT360     ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATA420     GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTT480     TTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCA540     AATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCG600     TCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCG660     ATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGA720     CGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGCTCTTATGCATGCTATACT780     GTTTTTGGCTTGGGCCAACACCCCGTCCTAGATAGGTGATGGTATAGCTTAGCCTATAGG840     TGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAA900     TCCATAACATGGCCGCTCTTTGCCACAACTATCTCTATTGGCTATATGCCAATACTCTGT960     CCTTCAGAGACTGACACGGACTCTGTATTTTTACAGGATGGGGTCCCATTTATTATTTAC1020     AAATTCACATATACAACAACGCCGTCCCCCGTGCCCGCAGTTTTTATTAACATGCGGGAT1080     CTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGC1140     TCCACATCCGAGCCTGTCCCATGCCCATGCCTCCAGCGGCTCATGGTCGCTCGGCAGCTC1200     CTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGGACAATGCCCACCACCACCAGTGT1260     GCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGAGATTGGGCTCG1320     CACCGCTGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGT1380     TGTTGTATTCTGTAGAGTTGGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCA1440     GTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGA1500     CTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGACACGATG1557     Met     GATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCA1605     AspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGlyAla     51015     GTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGC1653     ValPheValSerProSerGlnGluIleHisAlaArgPheArgArgGly     202530     GCCAGAACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTG1701     AlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValProVal     354045     TGGAAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCA1749     TrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLysAla     50556065     TATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTACCC1797     TyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysValPro     707580     ACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAAT1845     ThrAspProAsnProGlnGluValValLeuValAsnValThrGluAsn     859095     TTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATA1893     PheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAspIle     100105110     ATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACCCCA1941     IleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThrPro     115120125     CTCTGTGTTAGTTTAAAGTGCACTGATTTGGGGAATGCTACTAATACC1989     LeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsnThr     130135140145     AATAGTAGTAATACCAATAGTAGTAGCGGGGAAATGATGATGGAGAAA2037     AsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGluLys     150155160     GGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGAGGT2085     GlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArgGly     165170175     AAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATACCA2133     LysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIlePro     180185190     ATAGATAATGATACTACCAGCTATACGTTGACAAGTTGTAACACCTCA2181     IleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThrSer     195200205     GTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCCATA2229     ValIleThrGlnAlaCysProLysValSerPheGluProIleProIle     210215220225     CATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAG2277     HisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsnLys     230235240     ACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAATGT2325     ThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGlnCys     245250255     ACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTGAATGGC2373     ThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsnGly     260265270     AGTCTAGCAGAAGAAGAGGTAGTAATTAGATCTGCCAATTTCACAGAC2421     SerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThrAsp     275280285     AATGCTAAAACCATAATAGTACAGCTGAACCAATCTGTAGAAATTAAT2469     AsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIleAsn     290295300305     TGTACAAGACCCAACAACAATACAAGAAAAAGTATCCGTATCCAGAGG2517     CysThrArgProAsnAsnAsnThrArgLysSerIleArgIleGlnArg     310315320     GGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGA2565     GlyProGlyArgAlaPheValThrIleGlyLysIleGlyAsnMetArg     325330335     CAAGCACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTAAAA2613     GlnAlaHisCysAsnIleSerArgAlaLysTrpAsnAlaThrLeuLys     340345350     CAGATAGCTAGCAAATTAAGAGAACAATTTGGAAATAATAAAACAATA2661     GlnIleAlaSerLysLeuArgGluGlnPheGlyAsnAsnLysThrIle     355360365     ATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGT2709     IlePheLysGlnSerSerGlyGlyAspProGluIleValThrHisSer     370375380385     TTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTT2757     PheAsnCysGlyGlyGluPhePheTyrCysAsnSerThrGlnLeuPhe     390395400     AATAGTACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAATAAC2805     AsnSerThrTrpPheAsnSerThrTrpSerThrGluGlySerAsnAsn     405410415     ACTGAAGGAAGTGACACAATCACACTCCCATGCAGAATAAAACAATTT2853     ThrGluGlySerAspThrIleThrLeuProCysArgIleLysGlnPhe     420425430     ATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATC2901     IleAsnMetTrpGlnGluValGlyLysAlaMetTyrAlaProProIle     435440445     AGCGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACA2949     SerGlyGlnIleArgCysSerSerAsnIleThrGlyLeuLeuLeuThr     450455460465     AGAGATGGTGGTAATAACAACAATGGGTCCGAGATCTTCAGACCTGGA2997     ArgAspGlyGlyAsnAsnAsnAsnGlySerGluIlePheArgProGly     470475480     GGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAA3045     GlyGlyAspMetArgAspAsnTrpArgSerGluLeuTyrLysTyrLys     485490495     GTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGA3093     ValValLysIleGluProLeuGlyValAlaProThrLysAlaLysArg     500505510     AGAGTGGTGCAGAGAGAAAAATGAGCGGCCGC3125     ArgValValGlnArgGluLys     515520     (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 520 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyAlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuValAsnValThrGlu     859095     AsnPheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsn     130135140     ThrAsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGlu     145150155160     LysGlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArg     165170175     GlyLysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIle     180185190     ProIleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThr     195200205     SerValIleThrGlnAlaCysProLysValSerPheGluProIlePro     210215220     IleHisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsn     225230235240     LysThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGln     245250255     CysThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsn     260265270     GlySerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThr     275280285     AspAsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIle     290295300     AsnCysThrArgProAsnAsnAsnThrArgLysSerIleArgIleGln     305310315320     ArgGlyProGlyArgAlaPheValThrIleGlyLysIleGlyAsnMet     325330335     ArgGlnAlaHisCysAsnIleSerArgAlaLysTrpAsnAlaThrLeu     340345350     LysGlnIleAlaSerLysLeuArgGluGlnPheGlyAsnAsnLysThr     355360365     IleIlePheLysGlnSerSerGlyGlyAspProGluIleValThrHis     370375380     SerPheAsnCysGlyGlyGluPhePheTyrCysAsnSerThrGlnLeu     385390395400     PheAsnSerThrTrpPheAsnSerThrTrpSerThrGluGlySerAsn     405410415     AsnThrGluGlySerAspThrIleThrLeuProCysArgIleLysGln     420425430     PheIleAsnMetTrpGlnGluValGlyLysAlaMetTyrAlaProPro     435440445     IleSerGlyGlnIleArgCysSerSerAsnIleThrGlyLeuLeuLeu     450455460     ThrArgAspGlyGlyAsnAsnAsnAsnGlySerGluIlePheArgPro     465470475480     GlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeuTyrLysTyr     485490495     LysValValLysIleGluProLeuGlyValAlaProThrLysAlaLys     500505510     ArgArgValValGlnArgGluLys     515520     (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1532 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1522     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA48     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA96     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GGCGGCAGAGTAGAAAAGTTGTGGGTCACAGTCTATTATGGGGTACCT144     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     GTGTGGAAAGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAA192     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     GCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTA240     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     CCCACAGACCCCAACCCACAAGAAGTAGTATTGGAAAATGTAACAGAA288     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     CATTTTAACATGTGGAAAAATAACATGGTAGAACAGATGCAGGAGGAT336     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     ATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACC384     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     CCACTCTGTGTTACTTTAAATTGCAAGGATGTGAATGCTACTAATACC432     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ACTAATGATAGCGAGGGAACGATGGAGAGAGGAGAAATAAAAAACTGC480     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     TCTTTCAATATCACCACAAGCATAAGAGATGAGGTGCAGAAAGAATAT528     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     GCTCTTTTTTATAAACTTGATGTAGTACCAATAGATAATAATAATACC576     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     AGCTATAGGTTGATAAGTTGTGACACCTCAGTCATTACACAGGCCTGT624     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     CCAAAGATATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCT672     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GGTTTTGCGATTCTAAAGTGTAATGATAAGACGTTCAATGGAAAAGGA720     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     CCATGTAAAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCA768     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     GTAGTATCAACTCAACTGCTGCTAAATGGCAGTCTAGCAGAAGAAGAG816     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     GTAGTAATTAGATCTGACAATTTCACGAACAATGCTAAAACCATAATA864     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     GTACAGCTGAAAGAATCTGTAGAAATTAATTGTACAAGACCCAACAAC912     ValGlnLeuLysGluSerValGluIleAsnCysThrArgProAsnAsn     290295300     AATACAAGAAAAAGTATACATATAGGACCAGGGAGAGCATTTTATACT960     AsnThrArgLysSerIleHisIleGlyProGlyArgAlaPheTyrThr     305310315320     ACAGGAGAAATAATAGGAGATATAAGACAAGCACATTGTAACATTAGT1008     ThrGlyGluIleIleGlyAspIleArgGlnAlaHisCysAsnIleSer     325330335     AGAGCAAAATGGAATGACACTTTAAAACAGATAGTTATAAAATTAAGA1056     ArgAlaLysTrpAsnAspThrLeuLysGlnIleValIleLysLeuArg     340345350     GAACAATTTGAGAATAAAACAATAGTCTTTAATCACTCCTCAGGAGGG1104     GluGlnPheGluAsnLysThrIleValPheAsnHisSerSerGlyGly     355360365     GACCCAGAAATTGTAATGCACAGTTTTAATTGTGGAGGAGAATTTTTC1152     AspProGluIleValMetHisSerPheAsnCysGlyGlyGluPhePhe     370375380     TACTGTAATTCAACACAACTGTTTAATAGTACTTGGAATAATAATACT1200     TyrCysAsnSerThrGlnLeuPheAsnSerThrTrpAsnAsnAsnThr     385390395400     GAAGGGTCAAATAACACTGAAGGAAATACTATCACACTCCCATGCAGA1248     GluGlySerAsnAsnThrGluGlyAsnThrIleThrLeuProCysArg     405410415     ATAAAACAAATTATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTAT1296     IleLysGlnIleIleAsnMetTrpGlnGluValGlyLysAlaMetTyr     420425430     GCCCCTCCCATCAGAGGACAAATTAGATGTTCATCAAATATTACAGGG1344     AlaProProIleArgGlyGlnIleArgCysSerSerAsnIleThrGly     435440445     CTGCTATTAACAAGAGATGGTGGTATTAATGAGAATGGGACCGAGATC1392     LeuLeuLeuThrArgAspGlyGlyIleAsnGluAsnGlyThrGluIle     450455460     TTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTA1440     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     465470475480     TATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC1488     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     485490495     AAGGCAAAGAGAAGAGTGGTGCAAAGAGAAAAATGAGCGGCCGC1532     LysAlaLysArgArgValValGlnArgGluLys     500505     (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 507 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     ValGlnLeuLysGluSerValGluIleAsnCysThrArgProAsnAsn     290295300     AsnThrArgLysSerIleHisIleGlyProGlyArgAlaPheTyrThr     305310315320     ThrGlyGluIleIleGlyAspIleArgGlnAlaHisCysAsnIleSer     325330335     ArgAlaLysTrpAsnAspThrLeuLysGlnIleValIleLysLeuArg     340345350     GluGlnPheGluAsnLysThrIleValPheAsnHisSerSerGlyGly     355360365     AspProGluIleValMetHisSerPheAsnCysGlyGlyGluPhePhe     370375380     TyrCysAsnSerThrGlnLeuPheAsnSerThrTrpAsnAsnAsnThr     385390395400     GluGlySerAsnAsnThrGluGlyAsnThrIleThrLeuProCysArg     405410415     IleLysGlnIleIleAsnMetTrpGlnGluValGlyLysAlaMetTyr     420425430     AlaProProIleArgGlyGlnIleArgCysSerSerAsnIleThrGly     435440445     LeuLeuLeuThrArgAspGlyGlyIleAsnGluAsnGlyThrGluIle     450455460     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     465470475480     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     485490495     LysAlaLysArgArgValValGlnArgGluLys     500505     (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1484 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1474     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA48     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA96     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GGCGCCAGAACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCT144     GlyAlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     GTGTGGAAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAA192     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     GCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTA240     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     CCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAA288     ProThrAspProAsnProGlnGluValValLeuValAsnValThrGlu     859095     AATTTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGAT336     AsnPheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAsp     100105110     ATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACC384     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     CCACTCTGTGTTAGTTTAAAGTGCACTGATTTGGGGAATGCTACTAAT432     ProLeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsn     130135140     ACCAATAGTAGTAATACCAATAGTAGTAGCGGGGAAATGATGATGGAG480     ThrAsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGlu     145150155160     AAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGA528     LysGlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArg     165170175     GGTAAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATA576     GlyLysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIle     180185190     CCAATAGATAATGATACTACCAGCTATACGTTGACAAGTTGTAACACC624     ProIleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThr     195200205     TCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCC672     SerValIleThrGlnAlaCysProLysValSerPheGluProIlePro     210215220     ATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAAT720     IleHisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsn     225230235240     AAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAA768     LysThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGln     245250255     TGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTGAAT816     CysThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsn     260265270     GGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCTGCCAATTTCACA864     GlySerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThr     275280285     GACAATGCTAAAACCATAATAGTACAGCTGAACCAATCTGTAGAAATT912     AspAsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIle     290295300     AATTGTACAGGTGCTGGACATTGTAACATTAGTAGAGCAAAATGGAAT960     AsnCysThrGlyAlaGlyHisCysAsnIleSerArgAlaLysTrpAsn     305310315320     GCCACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAATTTGGAAAT1008     AlaThrLeuLysGlnIleAlaSerLysLeuArgGluGlnPheGlyAsn     325330335     AATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATT1056     AsnLysThrIleIlePheLysGlnSerSerGlyGlyAspProGluIle     340345350     GTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCA1104     ValThrHisSerPheAsnCysGlyGlyGluPhePheTyrCysAsnSer     355360365     ACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAA1152     ThrGlnLeuPheAsnSerThrTrpPheAsnSerThrTrpSerThrGlu     370375380     GGGTCAAATAACACTGAAGGAAGTGACACAATCACACTCCCATGCAGA1200     GlySerAsnAsnThrGluGlySerAspThrIleThrLeuProCysArg     385390395400     ATAAAACAATTTATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTAT1248     IleLysGlnPheIleAsnMetTrpGlnGluValGlyLysAlaMetTyr     405410415     GCCCCTCCCATCAGCGGACAAATTAGATGTTCATCAAATATTACAGGG1296     AlaProProIleSerGlyGlnIleArgCysSerSerAsnIleThrGly     420425430     CTGCTATTAACAAGAGATGGTGGTAATAACAACAATGGGTCCGAGATC1344     LeuLeuLeuThrArgAspGlyGlyAsnAsnAsnAsnGlySerGluIle     435440445     TTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTA1392     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     450455460     TATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC1440     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     465470475480     AAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAATGAGCGGCCGC1484     LysAlaLysArgArgValValGlnArgGluLys     485490     (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 491 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyAlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuValAsnValThrGlu     859095     AsnPheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsn     130135140     ThrAsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGlu     145150155160     LysGlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArg     165170175     GlyLysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIle     180185190     ProIleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThr     195200205     SerValIleThrGlnAlaCysProLysValSerPheGluProIlePro     210215220     IleHisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsn     225230235240     LysThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGln     245250255     CysThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsn     260265270     GlySerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThr     275280285     AspAsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIle     290295300     AsnCysThrGlyAlaGlyHisCysAsnIleSerArgAlaLysTrpAsn     305310315320     AlaThrLeuLysGlnIleAlaSerLysLeuArgGluGlnPheGlyAsn     325330335     AsnLysThrIleIlePheLysGlnSerSerGlyGlyAspProGluIle     340345350     ValThrHisSerPheAsnCysGlyGlyGluPhePheTyrCysAsnSer     355360365     ThrGlnLeuPheAsnSerThrTrpPheAsnSerThrTrpSerThrGlu     370375380     GlySerAsnAsnThrGluGlySerAspThrIleThrLeuProCysArg     385390395400     IleLysGlnPheIleAsnMetTrpGlnGluValGlyLysAlaMetTyr     405410415     AlaProProIleSerGlyGlnIleArgCysSerSerAsnIleThrGly     420425430     LeuLeuLeuThrArgAspGlyGlyAsnAsnAsnAsnGlySerGluIle     435440445     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     450455460     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     465470475480     LysAlaLysArgArgValValGlnArgGluLys     485490     (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1448 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1439     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA48     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA96     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GGCGGCAGAGTAGAAAAGTTGTGGGTCACAGTCTATTATGGGGTACCT144     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     GTGTGGAAAGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAA192     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     GCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTA240     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     CCCACAGACCCCAACCCACAAGAAGTAGTATTGGAAAATGTAACAGAA288     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     CATTTTAACATGTGGAAAAATAACATGGTAGAACAGATGCAGGAGGAT336     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     ATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACC384     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     CCACTCTGTGTTACTTTAAATTGCAAGGATGTGAATGCTACTAATACC432     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ACTAATGATAGCGAGGGAACGATGGAGAGAGGAGAAATAAAAAACTGC480     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     TCTTTCAATATCACCACAAGCATAAGAGATGAGGTGCAGAAAGAATAT528     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     GCTCTTTTTTATAAACTTGATGTAGTACCAATAGATAATAATAATACC576     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     AGCTATAGGTTGATAAGTTGTGACACCTCAGTCATTACACAGGCCTGT624     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     CCAAAGATATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCT672     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GGTTTTGCGATTCTAAAGTGTAATGATAAGACGTTCAATGGAAAAGGA720     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     CCATGTAAAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCA768     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     GTAGTATCAACTCAACTGCTGCTAAATGGCAGTCTAGCAGAAGAAGAG816     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     GTAGTAATTAGATCTGACAATTTCACGAACAATGCTAAAACCATAATA864     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     GTACAGCTGAAAGAATCTGTAGAAATTAATTGTACAGGTGCTGGACAT912     ValGlnLeuLysGluSerValGluIleAsnCysThrGlyAlaGlyHis     290295300     TGTAACATTAGTAGAGCAAAATGGAATGACACTTTAAAACAGATAGTT960     CysAsnIleSerArgAlaLysTrpAsnAspThrLeuLysGlnIleVal     305310315320     ATAAAATTAAGAGAACAATTTGAGAATAAAACAATAGTCTTTAATCAC1008     IleLysLeuArgGluGlnPheGluAsnLysThrIleValPheAsnHis     325330335     TCCTCAGGAGGGGACCCAGAAATTGTAATGCACAGTTTTAATTGTGGA1056     SerSerGlyGlyAspProGluIleValMetHisSerPheAsnCysGly     340345350     GGAGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGG1104     GlyGluPhePheTyrCysAsnSerThrGlnLeuPheAsnSerThrTrp     355360365     AATAATAATACTGAAGGGTCAAATAACACTGAAGGAAATACTATCACA1152     AsnAsnAsnThrGluGlySerAsnAsnThrGluGlyAsnThrIleThr     370375380     CTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGGAAGTAGGA1200     LeuProCysArgIleLysGlnIleIleAsnMetTrpGlnGluValGly     385390395400     AAAGCAATGTATGCCCCTCCCATCAGAGGACAAATTAGATGTTCATCA1248     LysAlaMetTyrAlaProProIleArgGlyGlnIleArgCysSerSer     405410415     AATATTACAGGGCTGCTATTAACAAGAGATGGTGGTATTAATGAGAAT1296     AsnIleThrGlyLeuLeuLeuThrArgAspGlyGlyIleAsnGluAsn     420425430     GGGACCGAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGG1344     GlyThrGluIlePheArgProGlyGlyGlyAspMetArgAspAsnTrp     435440445     AGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGA1392     ArgSerGluLeuTyrLysTyrLysValValLysIleGluProLeuGly     450455460     GTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAAAGAGAAAAATG1439     ValAlaProThrLysAlaLysArgArgValValGlnArgGluLys     465470475     AGCGGCCGC1448     (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 479 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     ValGlnLeuLysGluSerValGluIleAsnCysThrGlyAlaGlyHis     290295300     CysAsnIleSerArgAlaLysTrpAsnAspThrLeuLysGlnIleVal     305310315320     IleLysLeuArgGluGlnPheGluAsnLysThrIleValPheAsnHis     325330335     SerSerGlyGlyAspProGluIleValMetHisSerPheAsnCysGly     340345350     GlyGluPhePheTyrCysAsnSerThrGlnLeuPheAsnSerThrTrp     355360365     AsnAsnAsnThrGluGlySerAsnAsnThrGluGlyAsnThrIleThr     370375380     LeuProCysArgIleLysGlnIleIleAsnMetTrpGlnGluValGly     385390395400     LysAlaMetTyrAlaProProIleArgGlyGlnIleArgCysSerSer     405410415     AsnIleThrGlyLeuLeuLeuThrArgAspGlyGlyIleAsnGluAsn     420425430     GlyThrGluIlePheArgProGlyGlyGlyAspMetArgAspAsnTrp     435440445     ArgSerGluLeuTyrLysTyrLysValValLysIleGluProLeuGly     450455460     ValAlaProThrLysAlaLysArgArgValValGlnArgGluLys     465470475     (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1484 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1454     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA48     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA96     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GGCGCCAGAACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCT144     GlyAlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     GTGTGGAAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAA192     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     GCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTA240     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     CCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAA288     ProThrAspProAsnProGlnGluValValLeuValAsnValThrGlu     859095     AATTTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGAT336     AsnPheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAsp     100105110     ATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACC384     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     CCACTCTGTGTTAGTTTAAAGTGCACTGATTTGGGGAATGCTACTAAT432     ProLeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsn     130135140     ACCAATAGTAGTAATACCAATAGTAGTAGCGGGGAAATGATGATGGAG480     ThrAsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGlu     145150155160     AAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGA528     LysGlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArg     165170175     GGTAAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATA576     GlyLysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIle     180185190     CCAATAGATAATGATACTACCAGCTATACGTTGACAAGTTGTAACACC624     ProIleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThr     195200205     TCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCC672     SerValIleThrGlnAlaCysProLysValSerPheGluProIlePro     210215220     ATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAAT720     IleHisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsn     225230235240     AAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAA768     LysThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGln     245250255     TGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTGAAT816     CysThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsn     260265270     GGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCTGCCAATTTCACA864     GlySerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThr     275280285     GACAATGCTAAAACCATAATAGTACAGCTGAACCAATCTGTAGAAATT912     AspAsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIle     290295300     AATTGTACAGGTGCTGGACATTGTAACATTAGTAGAGCAAAATGGAAT960     AsnCysThrGlyAlaGlyHisCysAsnIleSerArgAlaLysTrpAsn     305310315320     GCCACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAATTTGGAAAT1008     AlaThrLeuLysGlnIleAlaSerLysLeuArgGluGlnPheGlyAsn     325330335     AATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATT1056     AsnLysThrIleIlePheLysGlnSerSerGlyGlyAspProGluIle     340345350     GTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCA1104     ValThrHisSerPheAsnCysGlyGlyGluPhePheTyrCysAsnSer     355360365     ACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAA1152     ThrGlnLeuPheAsnSerThrTrpPheAsnSerThrTrpSerThrGlu     370375380     GGGTCAAATAACACTGAAGGAAGTGACACAATCACACTCCCATGCAGA1200     GlySerAsnAsnThrGluGlySerAspThrIleThrLeuProCysArg     385390395400     ATAAAACAATTTATAAACATGGTGCAGGAAGTAGGAAAAGCAATGTAT1248     IleLysGlnPheIleAsnMetValGlnGluValGlyLysAlaMetTyr     405410415     GCCCCTCCCATCAGCGGACAAATTAGATGTTCATCAAATATTACAGGG1296     AlaProProIleSerGlyGlnIleArgCysSerSerAsnIleThrGly     420425430     CTGCTATTAACAAGAGATGGTGGTAATAACAACAATGGGTCCGAGATC1344     LeuLeuLeuThrArgAspGlyGlyAsnAsnAsnAsnGlySerGluIle     435440445     TTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTA1392     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     450455460     TATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC1440     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     465470475480     AAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAATGAGCGGCCGC1484     LysAlaLysArg     (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 484 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyAlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuValAsnValThrGlu     859095     AsnPheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsn     130135140     ThrAsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGlu     145150155160     LysGlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArg     165170175     GlyLysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIle     180185190     ProIleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThr     195200205     SerValIleThrGlnAlaCysProLysValSerPheGluProIlePro     210215220     IleHisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsn     225230235240     LysThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGln     245250255     CysThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsn     260265270     GlySerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThr     275280285     AspAsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIle     290295300     AsnCysThrGlyAlaGlyHisCysAsnIleSerArgAlaLysTrpAsn     305310315320     AlaThrLeuLysGlnIleAlaSerLysLeuArgGluGlnPheGlyAsn     325330335     AsnLysThrIleIlePheLysGlnSerSerGlyGlyAspProGluIle     340345350     ValThrHisSerPheAsnCysGlyGlyGluPhePheTyrCysAsnSer     355360365     ThrGlnLeuPheAsnSerThrTrpPheAsnSerThrTrpSerThrGlu     370375380     GlySerAsnAsnThrGluGlySerAspThrIleThrLeuProCysArg     385390395400     IleLysGlnPheIleAsnMetValGlnGluValGlyLysAlaMetTyr     405410415     AlaProProIleSerGlyGlnIleArgCysSerSerAsnIleThrGly     420425430     LeuLeuLeuThrArgAspGlyGlyAsnAsnAsnAsnGlySerGluIle     435440445     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     450455460     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     465470475480     LysAlaLysArg     (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1448 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1438     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA48     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA96     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GGCGGCAGAGTAGAAAAGTTGTGGGTCACAGTCTATTATGGGGTACCT144     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     GTGTGGAAAGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAA192     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     GCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTA240     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     CCCACAGACCCCAACCCACAAGAAGTAGTATTGGAAAATGTAACAGAA288     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     CATTTTAACATGTGGAAAAATAACATGGTAGAACAGATGCAGGAGGAT336     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     ATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACC384     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     CCACTCTGTGTTACTTTAAATTGCAAGGATGTGAATGCTACTAATACC432     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ACTAATGATAGCGAGGGAACGATGGAGAGAGGAGAAATAAAAAACTGC480     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     TCTTTCAATATCACCACAAGCATAAGAGATGAGGTGCAGAAAGAATAT528     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     GCTCTTTTTTATAAACTTGATGTAGTACCAATAGATAATAATAATACC576     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     AGCTATAGGTTGATAAGTTGTGACACCTCAGTCATTACACAGGCCTGT624     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     CCAAAGATATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCT672     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GGTTTTGCGATTCTAAAGTGTAATGATAAGACGTTCAATGGAAAAGGA720     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     CCATGTAAAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCA768     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     GTAGTATCAACTCAACTGCTGCTAAATGGCAGTCTAGCAGAAGAAGAG816     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     GTAGTAATTAGATCTGACAATTTCACGAACAATGCTAAAACCATAATA864     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     GTACAGCTGAAAGAATCTGTAGAAATTAATTGTACAGGTGCTGGACAT912     ValGlnLeuLysGluSerValGluIleAsnCysThrGlyAlaGlyHis     290295300     TGTAACATTAGTAGAGCAAAATGGAATGACACTTTAAAACAGATAGTT960     CysAsnIleSerArgAlaLysTrpAsnAspThrLeuLysGlnIleVal     305310315320     ATAAAATTAAGAGAACAATTTGAGAATAAAACAATAGTCTTTAATCAC1008     IleLysLeuArgGluGlnPheGluAsnLysThrIleValPheAsnHis     325330335     TCCTCAGGAGGGGACCCAGAAATTGTAATGCACAGTTTTAATTGTGGA1056     SerSerGlyGlyAspProGluIleValMetHisSerPheAsnCysGly     340345350     GGAGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGG1104     GlyGluPhePheTyrCysAsnSerThrGlnLeuPheAsnSerThrTrp     355360365     AATAATAATACTGAAGGGTCAAATAACACTGAAGGAAATACTATCACA1152     AsnAsnAsnThrGluGlySerAsnAsnThrGluGlyAsnThrIleThr     370375380     CTCCCATGCAGAATAAAACAAATTATAAACATGGTGCAGGAAGTAGGA1200     LeuProCysArgIleLysGlnIleIleAsnMetValGlnGluValGly     385390395400     AAAGCAATGTATGCCCCTCCCATCAGAGGACAAATTAGATGTTCATCA1248     LysAlaMetTyrAlaProProIleArgGlyGlnIleArgCysSerSer     405410415     AATATTACAGGGCTGCTATTAACAAGAGATGGTGGTATTAATGAGAAT1296     AsnIleThrGlyLeuLeuLeuThrArgAspGlyGlyIleAsnGluAsn     420425430     GGGACCGAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGG1344     GlyThrGluIlePheArgProGlyGlyGlyAspMetArgAspAsnTrp     435440445     AGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGA1392     ArgSerGluLeuTyrLysTyrLysValValLysIleGluProLeuGly     450455460     GTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAAAGAGAAAAAT1438     ValAlaProThrLysAlaLysArgArgValValGlnArgGluLys     465470475     GAGCGGCCGC1448     (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 479 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     ValGlnLeuLysGluSerValGluIleAsnCysThrGlyAlaGlyHis     290295300     CysAsnIleSerArgAlaLysTrpAsnAspThrLeuLysGlnIleVal     305310315320     IleLysLeuArgGluGlnPheGluAsnLysThrIleValPheAsnHis     325330335     SerSerGlyGlyAspProGluIleValMetHisSerPheAsnCysGly     340345350     GlyGluPhePheTyrCysAsnSerThrGlnLeuPheAsnSerThrTrp     355360365     AsnAsnAsnThrGluGlySerAsnAsnThrGluGlyAsnThrIleThr     370375380     LeuProCysArgIleLysGlnIleIleAsnMetValGlnGluValGly     385390395400     LysAlaMetTyrAlaProProIleArgGlyGlnIleArgCysSerSer     405410415     AsnIleThrGlyLeuLeuLeuThrArgAspGlyGlyIleAsnGluAsn     420425430     GlyThrGluIlePheArgProGlyGlyGlyAspMetArgAspAsnTrp     435440445     ArgSerGluLeuTyrLysTyrLysValValLysIleGluProLeuGly     450455460     ValAlaProThrLysAlaLysArgArgValValGlnArgGluLys     465470475     (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1571 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1567     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA48     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA96     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GGCGCCAGAACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCT144     GlyAlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     GTGTGGAAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAA192     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     GCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTA240     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     CCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAA288     ProThrAspProAsnProGlnGluValValLeuValAsnValThrGlu     859095     AATTTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGAT336     AsnPheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAsp     100105110     ATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACC384     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     CCACTCTGTGTTAGTTTAAAGTGCACTGATTTGGGGAATGCTACTAAT432     ProLeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsn     130135140     ACCAATAGTAGTAATACCAATAGTAGTAGCGGGGAAATGATGATGGAG480     ThrAsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGlu     145150155160     AAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGA528     LysGlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArg     165170175     GGTAAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATA576     GlyLysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIle     180185190     CCAATAGATAATGATACTACCAGCTATACGTTGACAAGTTGTAACACC624     ProIleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThr     195200205     TCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCC672     SerValIleThrGlnAlaCysProLysValSerPheGluProIlePro     210215220     ATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAAT720     IleHisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsn     225230235240     AAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAA768     LysThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGln     245250255     TGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTGAAT816     CysThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsn     260265270     GGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCTGCCAATTTCACA864     GlySerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThr     275280285     GACAATGCTAAAACCATAATAGTACAGCTGAACCAATCTGTAGAAATT912     AspAsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIle     290295300     AATTGTACAAGACCCAACAACAATACAAGAAAAAGTATCCGTATCCAG960     AsnCysThrArgProAsnAsnAsnThrArgLysSerIleArgIleGln     305310315320     AGGGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATG1008     ArgGlyProGlyArgAlaPheValThrIleGlyLysIleGlyAsnMet     325330335     AGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTA1056     ArgGlnAlaHisCysAsnIleSerArgAlaLysTrpAsnAlaThrLeu     340345350     AAACAGATAGCTAGCAAATTAAGAGAACAATTTGGAAATAATAAAACA1104     LysGlnIleAlaSerLysLeuArgGluGlnPheGlyAsnAsnLysThr     355360365     ATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCAC1152     IleIlePheLysGlnSerSerGlyGlyAspProGluIleValThrHis     370375380     AGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTG1200     SerPheAsnCysGlyGlyGluPhePheTyrCysAsnSerThrGlnLeu     385390395400     TTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAAT1248     PheAsnSerThrTrpPheAsnSerThrTrpSerThrGluGlySerAsn     405410415     AACACTGAAGGAAGTGACACAATCACACTCCCATGCAGAATAAAACAA1296     AsnThrGluGlySerAspThrIleThrLeuProCysArgIleLysGln     420425430     TTTATAAACATGGTGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCC1344     PheIleAsnMetValGlnGluValGlyLysAlaMetTyrAlaProPro     435440445     ATCAGCGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTA1392     IleSerGlyGlnIleArgCysSerSerAsnIleThrGlyLeuLeuLeu     450455460     ACAAGAGATGGTGGTAATAACAACAATGGGTCCGAGATCTTCAGACCT1440     ThrArgAspGlyGlyAsnAsnAsnAsnGlySerGluIlePheArgPro     465470475480     GGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATAT1488     GlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeuTyrLysTyr     485490495     AAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAG1536     LysValValLysIleGluProLeuGlyValAlaProThrLysAlaLys     500505510     AGAAGAGTGGTGCAGAGAGAAAAATGAGCGGCCGC1571     ArgArgValValGlnArgGluLys     515520     (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 520 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyAlaArgThrGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuValAsnValThrGlu     859095     AsnPheAsnMetTrpLysAsnAspMetValGluGlnMetHisGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValSerLeuLysCysThrAspLeuGlyAsnAlaThrAsn     130135140     ThrAsnSerSerAsnThrAsnSerSerSerGlyGluMetMetMetGlu     145150155160     LysGlyGluIleLysAsnCysSerPheAsnIleSerThrSerIleArg     165170175     GlyLysValGlnLysGluTyrAlaPhePheTyrLysLeuAspIleIle     180185190     ProIleAspAsnAspThrThrSerTyrThrLeuThrSerCysAsnThr     195200205     SerValIleThrGlnAlaCysProLysValSerPheGluProIlePro     210215220     IleHisTyrCysAlaProAlaGlyPheAlaIleLeuLysCysAsnAsn     225230235240     LysThrPheAsnGlyThrGlyProCysThrAsnValSerThrValGln     245250255     CysThrHisGlyIleArgProValValSerThrGlnLeuLeuLeuAsn     260265270     GlySerLeuAlaGluGluGluValValIleArgSerAlaAsnPheThr     275280285     AspAsnAlaLysThrIleIleValGlnLeuAsnGlnSerValGluIle     290295300     AsnCysThrArgProAsnAsnAsnThrArgLysSerIleArgIleGln     305310315320     ArgGlyProGlyArgAlaPheValThrIleGlyLysIleGlyAsnMet     325330335     ArgGlnAlaHisCysAsnIleSerArgAlaLysTrpAsnAlaThrLeu     340345350     LysGlnIleAlaSerLysLeuArgGluGlnPheGlyAsnAsnLysThr     355360365     IleIlePheLysGlnSerSerGlyGlyAspProGluIleValThrHis     370375380     SerPheAsnCysGlyGlyGluPhePheTyrCysAsnSerThrGlnLeu     385390395400     PheAsnSerThrTrpPheAsnSerThrTrpSerThrGluGlySerAsn     405410415     AsnThrGluGlySerAspThrIleThrLeuProCysArgIleLysGln     420425430     PheIleAsnMetValGlnGluValGlyLysAlaMetTyrAlaProPro     435440445     IleSerGlyGlnIleArgCysSerSerAsnIleThrGlyLeuLeuLeu     450455460     ThrArgAspGlyGlyAsnAsnAsnAsnGlySerGluIlePheArgPro     465470475480     GlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeuTyrLysTyr     485490495     LysValValLysIleGluProLeuGlyValAlaProThrLysAlaLys     500505510     ArgArgValValGlnArgGluLys     515520     (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1532 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..1522     (D) OTHER INFORMATION:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGA48     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     GCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA96     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GGCGGCAGAGTAGAAAAGTTGTGGGTCACAGTCTATTATGGGGTACCT144     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     GTGTGGAAAGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAA192     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     GCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTA240     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     CCCACAGACCCCAACCCACAAGAAGTAGTATTGGAAAATGTAACAGAA288     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     CATTTTAACATGTGGAAAAATAACATGGTAGAACAGATGCAGGAGGAT336     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     ATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACC384     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     CCACTCTGTGTTACTTTAAATTGCAAGGATGTGAATGCTACTAATACC432     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ACTAATGATAGCGAGGGAACGATGGAGAGAGGAGAAATAAAAAACTGC480     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     TCTTTCAATATCACCACAAGCATAAGAGATGAGGTGCAGAAAGAATAT528     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     GCTCTTTTTTATAAACTTGATGTAGTACCAATAGATAATAATAATACC576     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     AGCTATAGGTTGATAAGTTGTGACACCTCAGTCATTACACAGGCCTGT624     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     CCAAAGATATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCT672     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GGTTTTGCGATTCTAAAGTGTAATGATAAGACGTTCAATGGAAAAGGA720     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     CCATGTAAAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCA768     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     GTAGTATCAACTCAACTGCTGCTAAATGGCAGTCTAGCAGAAGAAGAG816     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     GTAGTAATTAGATCTGACAATTTCACGAACAATGCTAAAACCATAATA864     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     GTACAGCTGAAAGAATCTGTAGAAATTAATTGTACAAGACCCAACAAC912     ValGlnLeuLysGluSerValGluIleAsnCysThrArgProAsnAsn     290295300     AATACAAGAAAAAGTATACATATAGGACCAGGGAGAGCATTTTATACT960     AsnThrArgLysSerIleHisIleGlyProGlyArgAlaPheTyrThr     305310315320     ACAGGAGAAATAATAGGAGATATAAGACAAGCACATTGTAACATTAGT1008     ThrGlyGluIleIleGlyAspIleArgGlnAlaHisCysAsnIleSer     325330335     AGAGCAAAATGGAATGACACTTTAAAACAGATAGTTATAAAATTAAGA1056     ArgAlaLysTrpAsnAspThrLeuLysGlnIleValIleLysLeuArg     340345350     GAACAATTTGAGAATAAAACAATAGTCTTTAATCACTCCTCAGGAGGG1104     GluGlnPheGluAsnLysThrIleValPheAsnHisSerSerGlyGly     355360365     GACCCAGAAATTGTAATGCACAGTTTTAATTGTGGAGGAGAATTTTTC1152     AspProGluIleValMetHisSerPheAsnCysGlyGlyGluPhePhe     370375380     TACTGTAATTCAACACAACTGTTTAATAGTACTTGGAATAATAATACT1200     TyrCysAsnSerThrGlnLeuPheAsnSerThrTrpAsnAsnAsnThr     385390395400     GAAGGGTCAAATAACACTGAAGGAAATACTATCACACTCCCATGCAGA1248     GluGlySerAsnAsnThrGluGlyAsnThrIleThrLeuProCysArg     405410415     ATAAAACAAATTATAAACATGGTGCAGGAAGTAGGAAAAGCAATGTAT1296     IleLysGlnIleIleAsnMetValGlnGluValGlyLysAlaMetTyr     420425430     GCCCCTCCCATCAGAGGACAAATTAGATGTTCATCAAATATTACAGGG1344     AlaProProIleArgGlyGlnIleArgCysSerSerAsnIleThrGly     435440445     CTGCTATTAACAAGAGATGGTGGTATTAATGAGAATGGGACCGAGATC1392     LeuLeuLeuThrArgAspGlyGlyIleAsnGluAsnGlyThrGluIle     450455460     TTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTA1440     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     465470475480     TATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACC1488     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     485490495     AAGGCAAAGAGAAGAGTGGTGCAAAGAGAAAAATGAGCGGCCGC1532     LysAlaLysArgArgValValGlnArgGluLys     500505     (2) INFORMATION FOR SEQ ID NO:28:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 507 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGly     151015     AlaValPheValSerProSerGlnGluIleHisAlaArgPheArgArg     202530     GlyGlyArgValGluLysLeuTrpValThrValTyrTyrGlyValPro     354045     ValTrpLysGluAlaThrThrThrLeuPheCysAlaSerAspAlaLys     505560     AlaTyrAspThrGluValHisAsnValTrpAlaThrHisAlaCysVal     65707580     ProThrAspProAsnProGlnGluValValLeuGluAsnValThrGlu     859095     HisPheAsnMetTrpLysAsnAsnMetValGluGlnMetGlnGluAsp     100105110     IleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeuThr     115120125     ProLeuCysValThrLeuAsnCysLysAspValAsnAlaThrAsnThr     130135140     ThrAsnAspSerGluGlyThrMetGluArgGlyGluIleLysAsnCys     145150155160     SerPheAsnIleThrThrSerIleArgAspGluValGlnLysGluTyr     165170175     AlaLeuPheTyrLysLeuAspValValProIleAspAsnAsnAsnThr     180185190     SerTyrArgLeuIleSerCysAspThrSerValIleThrGlnAlaCys     195200205     ProLysIleSerPheGluProIleProIleHisTyrCysAlaProAla     210215220     GlyPheAlaIleLeuLysCysAsnAspLysThrPheAsnGlyLysGly     225230235240     ProCysLysAsnValSerThrValGlnCysThrHisGlyIleArgPro     245250255     ValValSerThrGlnLeuLeuLeuAsnGlySerLeuAlaGluGluGlu     260265270     ValValIleArgSerAspAsnPheThrAsnAsnAlaLysThrIleIle     275280285     ValGlnLeuLysGluSerValGluIleAsnCysThrArgProAsnAsn     290295300     AsnThrArgLysSerIleHisIleGlyProGlyArgAlaPheTyrThr     305310315320     ThrGlyGluIleIleGlyAspIleArgGlnAlaHisCysAsnIleSer     325330335     ArgAlaLysTrpAsnAspThrLeuLysGlnIleValIleLysLeuArg     340345350     GluGlnPheGluAsnLysThrIleValPheAsnHisSerSerGlyGly     355360365     AspProGluIleValMetHisSerPheAsnCysGlyGlyGluPhePhe     370375380     TyrCysAsnSerThrGlnLeuPheAsnSerThrTrpAsnAsnAsnThr     385390395400     GluGlySerAsnAsnThrGluGlyAsnThrIleThrLeuProCysArg     405410415     IleLysGlnIleIleAsnMetValGlnGluValGlyLysAlaMetTyr     420425430     AlaProProIleArgGlyGlnIleArgCysSerSerAsnIleThrGly     435440445     LeuLeuLeuThrArgAspGlyGlyIleAsnGluAsnGlyThrGluIle     450455460     PheArgProGlyGlyGlyAspMetArgAspAsnTrpArgSerGluLeu     465470475480     TyrLysTyrLysValValLysIleGluProLeuGlyValAlaProThr     485490495     LysAlaLysArgArgValValGlnArgGluLys     500505     (2) INFORMATION FOR SEQ ID NO:29:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 15 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     AlaProThrLysAlaLysArgArgValValGlnArgGluLysArg     151015     __________________________________________________________________________ 

What is claimed is:
 1. A mutant HIV-1 gp120 envelope glycoprotein comprising a V3 loop deletion and a C4 domain .sub.( w→X) point mutation, wherein X is an amino acid residue other than tryptophan, and wherein the mutations to the HIV-1 gp120 envelope glycoprotein reduce the affinity of the mutant HIV-1 gp120 envelope glycoprotein for CD4. 